Testis-specific cystatin-like protein cystatin T

ABSTRACT

The present invention relates to cystatin T (also known as zcys3) polypeptides and polynucleotides encoding the same. Cystatin T polypeptide is testis specific and homologous to cystatin-related epididymal specific gene (CRES) and type 2 cystatins. The polypeptides would be useful for modulating spermatogenesis and may be used to study or modulate that function in in vitro or in vivo systems. The present invention also includes antibodies to the cystatin T polypeptides.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. patent applicationSer. No. 09/431,480, filed on Nov. 1, 1999, which is related toProvisional Applications 60/109,217, filed on Nov. 20, 1998 and60/156,382 filed on Sep. 28, 1999. Under 35 U.S.C. §119(e) (1), thisapplication claims benefit of said Provisional Applications.

BACKGROUND OF THE INVENTION

The cystatin superfamily is an evolutionarily related group of proteinsconsisting of at least three families, i.e., stefins (type 1), cystatins(type 2), and kininogens (type 3). See, for example, Barrett, TIBS 12:193-196, 1987. Generally, stefin family members are unglycosylatedproteins consisting of about 100 amino acids that are devoid ofdisulfide bonds. In contrast, cystatin family members are proteinsconsisting of about 115 amino acids and characterized by two disulfidebonds in the carboxy-terminal region of the protein. Finally, kininogenscontain three regions containing two disulfide loops, similar to thecarboxy terminal domain found in members of the cystatin family. Thecystatin superfamily are inhibitors of cysteine proteinases (alsoreferred to as cysteine proteases) and are believed to function in aprotective role with regard to pathological action of endogenous orexogenous cysteine proteinases. It is believe that cystatins formequimolar reversible complexes with cysteine proteinases.

Cystatin-like proteins have also been identified. One such protein,cystatin-related epididymal specific gene (CRES) does not contain theconserved sequence motifs necessary for cysteine proteinase inhibitoryactivity (Cornwell et al., Mol. Endocrinol. 6:1653-64, 1992 and Cornwelland Hann, Mol. Reprod. Dev. 41:37-46, 1995). Also, unlike the ubiquitousexpression of many of the cystatins, CRES is restricted to the proximalcaput epididymal epithelium and testis. CRES expression is stagespecific during spermatogenesis and CRES is found in both round andelongating spermatids suggesting a specialized role duringspermatogenesis. Cystatins are also found with male reproductive tissuesand secretions. Cystatin C for example is found in highest abundance inhuman semen and participates in spermatogenesis and spermiogenesis andis associated with the sperm throughout its time in the male genitaltract (Esnard wt al., FEBS Lett. 300:131-5, 1992). Testatin is believedto be involved in early testis development. Expression is restricted tofetal gonads and adult testis and it is expressed during testis cordformation in pre-Sertoli cells (Töhönen et al., Proc. Natl. Acad. Sci.USA 95:14208-13, 1998).

Proteins capable of modulating spermatogenesis are sought for the study,diagnosis and treatment of conditions associated with reproductivedisorders. The present invention provides such polypeptides for theseand other uses that should be apparent to those skilled in the art fromthe teachings herein.

SUMMARY OF THE INVENTION

Within one aspect the invention provides an isolated polypeptidecomprising 10 or more contiguous amino acid residues of SEQ ID NO:2,wherein the polypeptide comprises SEQ ID NO:14 and specifically binds toan antibody to which a polypeptide of SEQ ID NO:2 specifically binds.Within one embodiment the polypeptide comprises SEQ ID NO:13. Withinanother embodiment the polypeptide comprises amino acid residues 76-138of SEQ ID NO:2. Within yet another embodiment the polypeptide furthercomprises an affinity tag or binding domain.

The invention also provides an isolated polypeptide comprising asequence of amino acid residues that is selected from the groupconsisting of: a) a sequence of amino acid residues that is 80%identical to the amino acid sequence of SEQ ID NO:2, wherein thesequence comprises cysteine residues corresponding to amino acidresidues 94, 104, 118 and 138 of SEQ ID NO:2 and wherein the polypeptidecomprising the amino acid sequence specifically binds with an antibodythat specifically binds with a polypeptide having the amino acidsequence of SEQ ID NO:2; and b) a sequence of amino acid residuesencoded by a polynucleotide sequence which hybridizes under stringentconditions to a similarly sized polynucleotide sequence of SEQ ID NO:1.Within one embodiment any difference between the amino acid sequence ofthe isolated polypeptide and the corresponding amino acid sequence ofSEQ ID NO:2 is due to a conservative amino acid substitution. Withinanother embodiment amino acid percent identity is determined using aFASTA program with ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=blosum62, with other parameters setas default.

The invention also provides an isolated polypeptide consisting of aminoacid residues 76-138 of SEQ ID NO:2. Additionally the invention providesan isolated polypeptide comprising the amino acid sequence of SEQ IDNO:2.

Within another aspect of the invention is provided a fusion proteincomprising a secretory signal sequence having the amino acid sequence ofamino acid residues 1-20 of SEQ ID NO:2, wherein the secretory signalsequence is operably linked to an additional polypeptide. In oneembodiment the first portion comprising a polypeptide as describedabove; and the second portion comprising another polypeptide.

Within yet another aspect of the invention is provided an isolatedpolynucleotide encoding a polypeptide as described above. Within oneembodiment the polypeptide comprises SEQ ID NO:13. Within anotherembodiment the polypeptide comprises amino acid residues 76-138 of SEQID NO:2. Within another embodiment the polynucleotide encodes apolypeptide further comprising an affinity tag or binding domain.

The invention also provides an isolated polynucleotide molecule thatencodes a polypeptide, wherein the polypeptide comprises a sequence ofamino acid residues that is selected from the group consisting of: a) asequence of amino acid residues that is 80% identical to the amino acidsequence of SEQ ID NO:2, wherein the sequence comprises cysteineresidues corresponding to amino acid residues 94, 104, 118 and 138 ofSEQ ID NO:2 and wherein the polypeptide comprising the amino acidsequence specifically binds with an antibody that specifically bindswith a polypeptide having the amino acid sequence of SEQ ID NO:2; b)degenerate nucleotide sequence of a); c) nucleotide sequencescomplementary to a) or b); and d) a sequence of amino acid residuesencoded by a polynucleotide sequence which hybridizes under stringentconditions to a similarly sized polynucleotide sequence of SEQ ID NO:1.Within one embodiment any difference between the amino acid sequenceencoded by the polynucleotide molecule and the corresponding amino acidsequence of SEQ ID NO:2 is due to one or more conservative amino acidsubstitutions. Within another embodiment the amino acid percent identityis determined using a FASTA program with ktup=1, gap opening penalty=10,gap extension penalty=1, and substitution matrix=blosum62, with otherparameters set as default.

The invention also provides an isolated polynucleotide moleculeconsisting of nucleotides 271-459 of SEQ ID NO:1. The invention furtherprovides an isolated polynucleotide molecule comprising the nucleotidesequence of nucleotides 46 to 468 of SEQ ID NO:1.

The invention provides a polynucleotide encoding a fusion proteincomprising a secretory signal sequence having the amino acid sequence ofamino acid residues 1-20 of SEQ ID NO:2, wherein the secretory signalsequence is operably linked to an additional polypeptide. Within oneembodiment the first portion comprises a polypeptide as described above;and the second portion comprising another polypeptide.

Within another aspect the invention provides an expression vectorcomprising the following operably linked elements: a transcriptionpromoter; a DNA segment encoding a polypeptide as described above; and atranscription terminator. Within one embodiment the expression vectorfurther comprises a secretory signal sequence operably linked to thepolypeptide encoded by the DNA segment. Within another aspect the DNAsegment encodes a polypeptide covalently linked amino terminally orcarboxy terminally to an affinity tag. Within yet another embodiment isprovided a cultured cell into which has been introduced an expressionvector as described above, wherein the cultured cell expresses thepolypeptide encoded by the DNA segment. Another embodiment provides amethod of producing a polypeptide comprising: culturing a cell intowhich has been introduced an expression vector as described above;whereby the cell expresses the polypeptide encoded by the DNA segment;and recovering the expressed polypeptide.

Within another aspect the invention provides an antibody or antibodyfragment that specifically binds to a polypeptide as described above.Within one embodiment the antibody is selected from the group consistingof: a) polyclonal antibody; b) murine monoclonal antibody; c) humanizedantibody derived from b); and d) human monoclonal antibody. Withinanother embodiment the antibody fragment is selected from the groupconsisting of F(ab′), F(ab), Fab′, Fab, Fv, scFv, and minimalrecognition unit. Within yet another embodiment is provided ananti-idiotype antibody that specifically binds to the antibody asdescribed above.

Within yet another aspect the invention provides a polypeptide asdescribed above, in combination with a pharmaceutically acceptablevehicle.

These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “variant” denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequence. The term allelic variant is also used herein to denote aprotein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides and proteins. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide or protein to denote proximity or relativeposition. For example, a certain sequence positioned carboxyl-terminalto a reference sequence within a polypeptide or protein is locatedproximal to the carboxyl terminus of the reference sequence, but is notnecessarily at the carboxyl terminus of the complete polypeptide orprotein.

The term “complements of polynucleotide molecules” denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” denotes a DNA molecule, linear or circular,that comprises a segment encoding a polypeptide of interest operablylinked to additional segments that provide for its transcription. Suchadditional segments may include promoter and terminator sequences, andmay optionally include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, and the like.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide molecule, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985). When applied to a polypeptide or protein, theterm “isolated” indicates that the polypeptide or protein is found in acondition other than its native environment, such as apart from bloodand animal tissue. In a preferred form, the isolated polypeptide orprotein is substantially free of other proteins, particularly otherproteins of animal origin. It is preferred to provide the protein in ahighly purified form, i.e., greater than 95% pure, more preferablygreater than 99% pure. When used in this context, the term “isolated”does not exclude the presence of the same polypeptide in alternativephysical forms, such as dimers or alternatively glycosylated orderivatized forms.

The term “operably linked”, when referring to DNA segments, denotes thatthe segments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in the promoter andproceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, a-globin, b-globin, and myoglobin are paralogs of each other.

The term “polynucleotide” denotes a single- or double-stranded polymerof deoxyribonucleotide or ribonucleotide bases read from the 5′ to the3′ end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

“Probes and/or primers” as used herein can be RNA or DNA. DNA can beeither cDNA or genomic DNA. Polynucleotide probes and primers are singleor double-stranded DNA or RNA, generally synthetic oligonucleotides, butmay be generated from cloned cDNA or genomic sequences or itscomplements. Analytical probes will generally be at least 20 nucleotidesin length, although somewhat shorter probes (14-17 nucleotides) can beused. PCR primers are at least 5 nucleotides in length, preferably 15 ormore nt, more preferably 20-30 nt. Short polynucleotides can be usedwhen a small region of the gene is targeted for analysis. For grossanalysis of genes, a polynucleotide probe may comprise an entire exon ormore. Probes can be labeled to provide a detectable signal, such as withan enzyme, biotin, a radionuclide, fluorophore, chemiluminescer,paramagnetic particle and the like, which are commercially availablefrom many sources, such as Molecular Probes, Inc., Eugene, Oreg., andAmersham Corp., Arlington Heights, Ill., using techniques that are wellknown in the art.

The term “promoter” denotes a portion of a gene containing DNA sequencesthat provide for the binding of RNA polymerase and initiation oftranscription. Promoter sequences are commonly, but not always, found inthe 5′ non-coding regions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. Most nuclear receptors also exhibit amulti-domain structure, including an amino-terminal, transactivatingdomain, a DNA binding domain and a ligand binding domain. In general,receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g.,thyroid stimulating hormone receptor, beta-adrenergic receptor) ormultimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor,GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part upon the discovery of a novel DNAsequence that encodes a cystatin T (also known as zcys3), polypeptidehaving homology to the cystatin family of proteins (i.e., cystatinsuperfamily type 2 proteins). Indicia of such homology are the fourcysteine residues in the carboxy terminal portion of the protein (atpositions 94, 104, 118 and 138 of SEQ ID NO: 2), which are believed toform two disulfide bonds thereby generating the characteristic twodisulfide loop structure of the cystatin family.

Novel cystatin T polypeptides of the present invention were initiallyidentified by selecting ESTs from an EST database and predicting theprotein sequences thereof. Selected EST/corresponding protein sequenceswere associated with the most similar functionally characterizedpolynucleotide or polypeptide sequence using indexing software. ESTshomologous to secreted proteins having interesting associated biologicalactivities were selected for further study. A single EST sequence wasdiscovered and predicted to be a mouse paralogue of the murinecystatin-related epididymal specific protein precursor CRES (SEQ IDNO:3), murine Testatin (SEQ ID NO:18), human cystatin C precursor (SEQID NO:6), cystatin D precursor (SEQ ID NO:7) and cystatin SN precursor(CYTN_HUMAN) known cysteine proteinase inhibitors of the cystatin family(i.e., cystatin superfamily type 2). See, for example, Abrahamson etal., FEBS Lett. 216 229-33, 1987.

When compared with other members of the cystatin family, especiallyhuman and mouse CRES, the cystatin T polypeptides were found to containa motif QX(6)YX(10)CXKX(7-12)CX(13)CX(7)PWX(10)C (SEQ ID NO:13) locatedat amino acid residues 76-138 of SEQ ID NO:2, wherein X( ) representsthe number of amino acid residues between the identified amino acidresidues. X represents any amino acid residue, Q represents glutamine, Yrepresents tyrosine, C represents cysteine, K represents lysine, Prepresents proline and W represents tryptophan. Within this motif is acysteine motif that appears to be unique to cystatin superfamilyproteins. The cysteine motif is represented by the formula CX{8, 9 or10}CX{13}CX{19}C, (SEQ ID NO:14) wherein X{ } is the number of aminoacid residues between cysteines (C). This motif can be found at aminoacid residues 94-138 of SEQ ID NO:2. This cysteine motif appears tooccur in all known members of the cystatin family (for example, humancystatins C, S, T, N, D, bovine cystatin, chick cystatin, rat cystatinsC and S, mouse cystatin C, Testatin and the like).

X-ray analysis of chicken egg white cystatin (Bode et al., EMBO J. 7,2593-99, 1988) and directed mutagenesis studies (Auerswald et al., Eur.J. Biochem. 209:837-45, 1992) revealed three regions to be important forcysteine proteinase inhibitory activity, the Gly9 residue, the Gln53,Val55, Gly57 motif, and the carboxy terminal Pro103, Trp104 motif. Onlythe Pro103, Trp104 motif and the Gln53 residue are conserved in CRESwhile Testatin retains only the Val55 and Pro103, Trp104 motif. It isnot clear if the substrate specificity of these members is altered or ifthese proteins lack the ability to ihibit cysteine proteinases. Töhönenet al. Proc. Natl. Acad. Sci. USA 95:14208-13, 1998, propose that thesetwo genes comprise a subgroup within the family 2 cysteins. Cystatin Talso contains a subset of these motif, having the only the Gln53 (aminoacid residue 76 of SEQ ID NO:2) residue of the Gln53, Val55, Gly57region although within this region (amino acid residues 76-79 of SEQ IDNO:2) the four residues are identical to those present in CRES. CystatinT also contains the Pro103, Trp104 (amino acid residues 126-137 of SEQID NO:2) and a Gly residue near the amino terminus (amino acid residue76 of SEQ ID NO:2) but the spacing is not consistent with that observedfor other family members. These similarities between cystatin T, CRESand Testatin suggest they form a new family 2 subclass.

Cystatin T links to murine chromosome 2 framework marker d2mit194located at 81.4 cm. The human locus for this position is 20p11.2, whichcontains the cystatin gene cluster (five cystatin genes, CST1 to 5, andtwo pseudogenes, Thiesse et al., DNA Cell Biol. 13:97-116, 1994) thatspans less than 905 kb. Members of family 1 and 3 have been mapped tothe long arm of chromosome 3 (Tsui et al., Genomics 15:507-14, 1993, andJames et al., Genomics 32:425-30, 1996) further suggesting that cystatinT is a member of the family 2 cystatins.

Analysis of the tissue distribution of the mRNA corresponding to thisnovel DNA by both Northern blot and Dot blot showed selective expressionin murine testis. One transcript size was observed at approximately 1.0Kb. The polypeptide encoded by that polynucleotide sequence has beendesignated cystatin T. Human tissue Northern blots probed with murinesequence indicated expression of a 1.0 Kb transcript in trachea.Cystatin T expression is quite distinct from other family 2 cystatinswith the exception of CRES and Testatin. CRES is found primarily in theproximal caput region of the epididymis in addition to low expression intestis (Cornwell et al., Mol. Endo. 6:1653-64, 1992). Testatinexpression is restricted to pre-Sertoli cells in fetal tissue and toSertioli cells in adult testis (Töhönen et al., ibid.). Cystatin T mRNAexpression is high in primary testis, it was not detected in the testiscell lines tested which may suggest that like CRES (Cornwell et al.,ibid.) it is dependent on a various hormones in the testis, or is notexpressed in these cell lines.

Analysis of the DNA encoding a cystatin T polypeptide (SEQ ID NO:1)revealed an open reading frame encoding 141 amino acids (SEQ ID NO: 2)comprising a signal peptide of 20 amino acid residues (residues 1-20 ofSEQ ID NO: 2, nucleotides 57-105 of SEQ ID NO:1) and a maturepolypeptide of 121 amino acids (residues 21-141 of SEQ ID NO: 2,nucleotides 106-468 of SEQ ID NO:1). Those skilled in the art willrecognize that predicted secretory signal sequence domain boundaries areapproximations based on primary sequence content, and may vary slightly;however, such estimates are generally accurate to within ±4 amino acidresidues.

It is generally believed that under selective pressure for organisms toacquire new biological functions new cystatin superfamily members arosefrom duplication of existing receptor genes leading to the existence ofmulti-gene families. Family members thus contain vestiges of theancestral gene, and these characteristic features can be exploited inthe isolation and identification of additional family members. Thecystatin superfamily is subdivided into stefins, cystatins andkininogens. Stefins are single chain proteins of M_(r) of about 11,000that lack disulfide bonds and carbohydrates. Cystatins are single chainproteins characterized by the presence of two disulfide bonds in thecarboxy terminal region thereof. Moreover, cystatins are composed ofabout 115 amino acids with M_(r) of about 13,000. Some cystatins exhibitphosphorylated and dephosphorylated forms. Kininogens are highermolecular weight moieties that contain three regions of homology tocystatins (i.e., three sets of two disulfide loops); however, only twoof such regions are believed to be active. See, for example, Turk andBode, FEBS Lett. 285(2): 213-9, 1991; Barrett, TIBS 12: 193-6, 1987; andBobek and Levine, Crit. Rev. Oral Biol. Med. 3: 307-32, 1992.

Cystatin T shares 37.1% identity at the amino acid level with bothmurine (SEQ ID NO:3) and human (SEQ ID NO:4) CRES (Cornwall et al., Mol.Endocrinol. 6:1653-64, 1992). Cystatin T is slightly less identical toother members of the cystatin family. At the amino acid level, CystatinT shares 28.3% identity with cystatin C_mouse (Solem et al., Biochem.Biophys. Res. Comm. 172:945-51, 1990, SEQ ID NO:5); 30.0% identity withcystatin C_HUMAN (Abrahamson et al., FEBS Lett. 216(2): 229-33, 1987,SEQ ID NO:6); 27.2% identity with cystatin D_HUMAN (Freije et al., J.Biol. Chem. 266(30): 20538-43, 1991, SEQ ID NO:7); 24.0% identity withcystatin E_HUMAN (Ni et al., J. Biol. Chem. 272:10853-8, 1997, SEQ IDNO:8); 23.8% identity with cystatin F_HUMAN (Ni et al., J. Biol. Chem.273:24797-804, 1998, SEQ ID NO:9); 25.3% identity with cystatin M_HUMAN(Sotiropoulou et al., J. Biol. Chem. 373:903-10, 1997, SEQ ID NO:10);26.9% identity with cystatin S_HUMAN (Bobek et al., Biochem. J. 278:627-35, 1991, SEQ ID NO:11), 28.3% identity with cystatin SA-I_HUMAN(Al-Hashim et al., J. Biol. Chem. 263:9381-87, 1988, SEQ ID NO:12) and18.8% identity with Testatin (Töhönen et al., Proc. Natl. Acad. Sci.USA.95:14208-13, 1998).

Regulation of reproductive function in males and females is controlledin part by feedback inhibition of the hypothalamus and anteriorpituitary by blood-bone hormones. Testis proteins, such as activins andinhibins, have been shown to regulate secretion of active moleculesincluding follicle stimulating hormone (FSH) for the pituitary (Ying,Endocr. Rev. 9:267-93, 1988; Plant et al., Hum. Reprod. 8:41-44, 1993).CRES gene expression is thought to be regulated by testicular factors orhormones (Cornwall et al., Mol. Endo. 6:1653-64, 1992). These functionsmay also be associated with the cystatin T proteins and may be used toregulate testicular functions.

Spermatogenesis is the sequential process whereby germ cells ultimatelymature into spermatozoa, herein referred to as sperm. Testis-specificfactors that influence the maturation process may come directly from theSertoli cells that are in contact with spermatogenic cells, or may beparacrine or endocrine factors. Many of the molecules produced outsidethe seminiferous tubules are transported into the sperm cellmicroenvironment by transport and binding proteins that are expressed bythe Sertoli cells within the seminiferous tubules.

Paracrine factors that cross the cellular barrier and enter the spermcell microenvironment include molecules secreted from cells Leydigcells. Leydig cells are located in the interstitial space found betweenthe seminiferous tubules in the testis, and produce several factorsbelieved to play an important role in spermatogenic cell maturationprocess, such as testosterone, Leydig factor, IGF-1, inhibin andactivin. The expression of these, and other factors, may be specific toa defined stage in the spermatogenic cycle. Cystatin T expression wasnot detected in epididymus or seminal vesicles suggesting that it mightbe expressed by interstitial cells.

Molecules acting in the early stages of spermatogenesis may be involvedin such activities as sperm proliferation and development. Suchmolecules could act to enhance sperm development. CRES expression isstage-specific during spermatogenesis and is exclusively expressed byround spermatids at Stages VII-VIII and by early elongating spermatidsof Stages IX and X (Cornwall and Hann, Mol. Reprod. Dev. 41:37-46,1995).

Those molecules acting at a later stage in spermatogenesis may beinvolved in sperm motility and egg-sperm interactions. Such moleculescould also act to enhance the activity of cryopreserved sperm. Assaysevaluating such activities are known (Rosenberger, J. Androl. 11:89-96,1990; Fuchs, Zentralbl Gynakol 11:117-20, 1993; Neurwinger et al.,Andrologia 22:335-9, 1990; Harris et al., Hum. Reprod. 3:856-60, 1988;and Jockenhovel, Andrologia 22:171-8, 1990; Lessing et al., Fertil.Steril. 44:406-9 (1985); Zaneveld, In Male Infertility Chapter 11,Comhaire Ed., Chapman & Hall, London 1996).

Proteolysis-regulated testis-specific functions, including regulation ofinteractions between various cell types in the seminiferous tubuleduring spermatogenesis as well as migration of germ cells and release ofspermatids (Monsees et al., Adv. Exp. Med. Biol. 424:111-23, 1997),suggest a role for the cystatins in the male reproduction process.Cysteine proteinases, such as cathepsin L, are present in latent form inthe spermatozoa. Inhibitors of cysteine proteinases, such as cystatin Cwhich is found in abundance in testis, epididymis, prostate and seminalvesicles may play a role in controlling proteolytic activity.Cystatin-like proteins such as cystatin T which have alterations insequences thought to be necessary for inhibition of known proteinasesmay serve to inhibit as yet unidentified proteinases and could alsoserve to modulate proteinase activity in the testis. Cystatin T mayregulate testis-specific cysteine proteinases such as testis-thymusexpressed cathepsin V (Brömme et al., Biochem. 38:2377-85, 1999).Molecules having such activities are expected to result in enhancedfertility and successful reproduction. Antagonists of such moleculeswould be useful in contraceptive applications.

Proteins of the present invention would find application in enhancingfertilization during assisted reproduction in humans and in animals.Such assisted reproduction methods are known in the art and includeartificial insemination, in vitro fertilization, embryo transfer andgamete intrafallopian transfer. Such methods are useful for assistingmen and women who may have physiological or metabolic disorders thatprevent natural conception. Such methods are also useful in animalbreeding programs, such as for livestock, zoological, endangered speciesor racehorse breeding, and could be used within methods for the creationof transgenic animals.

Proteins of the present invention can be used to enhance spermproduction, to increase the number of viable sperm in a sample, or becombined with sperm, an egg or an egg-sperm mixture prior tofertilization of the egg to enhance fertilization. The washed sperm orsperm removed from the seminal plasma used in such assisted reproductionmethods has been shown to have altered reproductive functions, inparticular, reduced motility and zona interaction. To enhancefertilization during assisted reproduction methods sperm is capacitatedusing exogenously added compounds. Suspension of the sperm in seminalplasma from normal subjects or in a “capacitation media” containing acocktail of compounds known to activate sperm, such as caffeine, dibutylcyclic adenosine monophosphate (dbcAMP) or theophylline, have resultedin improved reproductive function of the sperm, in particular, spermmotility and zonae penetration (Park et al., Am. J. Obstet. Gynecol.158:974-9, 1988; Vandevoort et al., Mol. Repro. Develop. 37:299-304,1993; Vandevoort and Overstreet, J. Androl. 16:327-33, 1995).Polypeptides of the present invention can used in such methods toenhance viability of cryopreserved sperm, enhance sperm motility andenhance fertilization, particularly in association with methods ofassisted reproduction.

In cases where pregnancy is not desired, cystatin T proteins or proteinfragments may function as germ-cell-specific antigens for use ascomponents in “immunocontraceptive” or “anti-fertility” vaccines toinduce formation of antibodies and/or cell mediated immunity toselectively inhibit a process, or processes, critical to successfulreproduction in humans and animals. The use of sperm and testis antigensin the development of an immunocontraceptive have been described (O'Hernet al., Biol Reprod. 52:311-39, 1995; Diekman and Herr, Am. J. Reprod.Immunol. 37:111-17, 1997; Zhu and Naz, Proc. Natl. Acad. Sci. USA94:4704-9,1997). A vaccine based on human chorionic gonadotrophin (HCG)linked to a diphtheria or tetanus carrier is currently in clinicaltrials (Talwar et al., Proc. Natl. Acad. Sci. USA 91:8532-36, 1994). Asingle injection resulted in production of high titer antibodies thatpersisted for nearly a year in rabbits (Stevens, Am. J. Reprod. Immunol.29:176-88, 1993). Such methods of immunocontraception using vaccineswould include testis specific proteins such as cystatin T or fragmentthereof. The cystatin T protein or fragments can be conjugated to acarrier protein or peptide, such as tetanus or diphtheria toxoid. Anadjuvant, as described above, can be included and the protein orfragment can be noncovalently associated with other molecules to enhanceintrinsic immunoreactivity. Methods for administration and methods fordetermining the number of administrations are known in the art. Such amethod might include a number of primary injections over several weeksfollowed by booster injections as needed to maintain a suitable antibodytiter.

The present invention also provides polynucleotide molecules, includingDNA and RNA molecules, that encode the cystatin T polypeptides disclosedherein. Those skilled in the art will readily recognize that, in view ofthe degeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules. SEQ ID NO:15 is adegenerate DNA sequence that encompasses all DNAs that encode thecystatin T polypeptide of SEQ ID NO:2. Those skilled in the art willrecognize that the degenerate sequence of SEQ ID NO:15 also provides allRNA sequences encoding SEQ ID NO:2 by substituting U (uracil) for T(thymine). Thus, cystatin T polypeptide-encoding polynucleotidescomprising nucleotide 1 to nucleotide 423 of SEQ ID NO:15 and their RNAequivalents are contemplated by the present invention. Table 1 setsforth the one-letter codes used within SEQ ID NO:15 to denote degeneratenucleotide positions. “Resolutions” are the nucleotides denoted by acode letter. “Complement” indicates the code for the complementarynucleotide(s). For example, the code Y denotes either C (cytosine) or T,and its complement R denotes A (adenine) or G (guanine), A beingcomplementary to T, and G being complementary to C.

TABLE 1 Nucleotide Resolution Nucleotide Complement A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:15, encompassing all possiblecodons for a given amino acid, are set forth in Table 2.

TABLE 2 One Degen- Amino Letter erate Acid Code Codons Codon Cys C TGCTGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN ProP CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGTGGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAGCAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAGAAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTGYTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp WTGG TGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:2. Variant sequences can be readily tested forfunctionality as described herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit “preferential codon usage.” In general, see,Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:15 serves as a template for optimizing expression of polynucleotidesin various cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

The highly conserved amino acids, both within and without the region ofhigh identity, can be used as a tool to identify cystatin T polypeptidesor cystatin T-like proteins. For instance, reversetranscription-polymerase chain reaction (RT-PCR) can be used to amplifysequences encoding the conserved motifs suggested by the multiplealignment from RNA obtained from a variety of tissue sources. Inparticular, one such probe would include a polynucleotide sequenceencoding the amino acid sequence of amino acid residues 95-104 of SEQ IDNO:2. Also included in this aspect of the present invention arepolypeptide probes.

Nucleic acid molecules disclosed herein can be used to detect theexpression of a cystatin T gene in a biological sample. Such probemolecules include double-stranded nucleic acid molecules comprising thenucleotide sequences of SEQ ID NOs:1 or 15, or fragments thereof, aswell as single-stranded nucleic acid molecules having the complement ofthe nucleotide sequences of SEQ ID NOs:1 or 15, or a fragment thereof.Probe molecules may be DNA, RNA, oligonucleotides, and the like.

As an illustration, suitable probes include nucleic acid molecules thatbind with a portion of a cystatin T domain or motif, such as thecystatin T cysteine motif (nucleotides 325-459 of SEQ ID NO:1 ornucleotides 280-414 of SEQ ID NO:15). Other probes include nucleotides271-459 of SEQ ID NO:1 or nucleotides 226-414 of SEQ ID NO:15.

In a basic assay, a single-stranded probe molecule is incubated withRNA, isolated from a biological sample, under conditions of temperatureand ionic strength that promote base pairing between the probe andtarget cystatin T RNA species. After separating unbound probe fromhybridized molecules, the amount of hybrids is detected.

Well-established hybridization methods of RNA detection include northernanalysis and dot/slot blot hybridization (see, for example, Ausubelibid. and Wu et al. (eds.), “Analysis of Gene Expression at the RNALevel,” in Methods in Gene Biotechnology, pages 225-239 (CRC Press, Inc.1997)). Nucleic acid probes can be detectably labeled with radioisotopessuch as ³²P or ³⁵S. Alternatively, cystatin T RNA can be detected with anonradioactive hybridization method (see, for example, Isaac (ed.),Protocols for Nucleic Acid Analysis by Nonradioactive Probes, HumanaPress, Inc., 1993). Typically, nonradioactive detection is achieved byenzymatic conversion of chromogenic or chemiluminescent substrates.Illustrative nonradioactive moieties include biotin, fluorescein, anddigoxigenin.

Cystatin T oligonucleotide probes are also useful for in vivo diagnosis.As an illustration, ¹⁸F-labeled oligonucleotides can be administered toa subject and visualized by positron emission tomography (Tavitian etal., Nature Medicine 4:467, 1998).

Numerous diagnostic procedures take advantage of the polymerase chainreaction (PCR) to increase sensitivity of detection methods. Standardtechniques for performing PCR are well-known (see, generally, Mathew(ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),White (ed.), PCR Protocols: Current Methods and Applications (HumanaPress, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (HumanaPress, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (HumanaPress, Inc. 1998)). PCR primers can be designed to amplify a sequenceencoding a particular cystatin T domain or motif, such as the cystatin Tcysteine motif (encoded by about nucleotide 325-459 of SEQ ID NO:1 ornucleotides 280-414 of SEQ ID NO:15).

One variation of PCR for diagnostic assays is reverse transcriptase-PCR(RT-PCR). In the RT-PCR technique, RNA is isolated from a biologicalsample, reverse transcribed to cDNA, and the CDNA is incubated withcystatin T primers (see, for example, Wu et al. (eds.), “Rapid Isolationof Specific cDNAs or Genes by PCR,” in Methods in Gene Biotechnology,CRC Press, Inc., pages 15-28, 1997). PCR is then performed and theproducts are analyzed using standard techniques.

As an illustration, RNA is isolated from biological sample using, forexample, the guanidinium-thiocyanate cell lysis procedure describedherein. Alternatively, a solid-phase technique can be used to isolatemRNA from a cell lysate. A reverse transcription reaction can be primedwith the isolated RNA using random oligonucleotides, short homopolymersof dT, or cystatin T anti-sense oligomers. Oligo-dT primers offer theadvantage that various mRNA nucleotide sequences are amplified that canprovide control target sequences. Cystatin T sequences are amplified bythe polymerase chain reaction using two flanking oligonucleotide primersthat are typically at least 5 bases in length.

PCR amplification products can be detected using a variety ofapproaches. For example, PCR products can be fractionated by gelelectrophoresis, and visualized by ethidium bromide staining.Alternatively, fractionated PCR products can be transferred to amembrane, hybridized with a detectably-labeled cystatin T probe, andexamined by autoradiography. Additional alternative approaches includethe use of digoxigenin-labeled deoxyribonucleic acid triphosphates toprovide chemiluminescence detection, and the C-TRAK colorimetric assay.

Another approach is real time quantitative PCR (Perkin-Elmer Cetus,Norwalk, Conn.). A fluorogenic probe, consisting of an oligonucleotidewith both a reporter and a quencher dye attached, anneals specificallybetween the forward and reverse primers. Using the 5′ endonucleaseactivity of Taq DNA polymerase, the reporter dye is separated from thequencher dye and a sequence-specific signal is generated and increasesas amplification increases. The fluorescence intensity can becontinuously monitored and quantified during the PCR reaction.

Another approach for detection of cystatin T expression is cycling probetechnology (CPT), in which a single-stranded DNA target binds with anexcess of DNA-RNA-DNA chimeric probe to form a complex, the RNA portionis cleaved with RNase H, and the presence of cleaved chimeric probe isdetected (see, for example, Beggs et al., J. Clin. Microbiol. 34:2985,1996 and Bekkaoui et al., Biotechniques 20:240, 1996). Alternativemethods for detection of cystatin T sequences can utilize approachessuch as nucleic acid sequence-based amplification (NASBA), cooperativeamplification of templates by cross-hybridization (CATCH), and theligase chain reaction (LCR) (see, for example, Marshall et al., U.S.Pat. No. 5,686,272 (1997), Dyer et al., J. Virol. Methods 60:161, 1996;Ehricht et al., Eur. J. Biochem. 243:358, 1997 and Chadwick et al., J.Virol. Methods 70:59, 1998). Other standard methods are known to thoseof skill in the art.

Cystatin T probes and primers can also be used to detect and to localizecystatin T gene expression in tissue samples. Methods for such in situhybridization are well-known to those of skill in the art (see, forexample, Choo (ed.), In Situ Hybridization Protocols, Humana Press,Inc., 1994; Wu et al. (eds.), “Analysis of Cellular DNA or Abundance ofmRNA by Radioactive In Situ Hybridization (RISH),” in Methods in GeneBiotechnoloqy, CRC Press, Inc., pages 259-278, 1997 and Wu et al.(eds.), “Localization of DNA or Abundance of mRNA by Fluorescence InSitu Hybridization (RISH),” in Methods in Gene Biotechnology, CRC Press,Inc., pages 279-289, 1997).

Various additional diagnostic approaches are well-known to those ofskill in the art (see, for example, Mathew (ed.), Protocols in HumanMolecular Genetics Humana Press, Inc., 1991; Coleman and Tsongalis,Molecular Diagnostics, Humana Press, Inc., 1996 and Elles, MolecularDiagnosis of Genetic Diseases, Humana Press, Inc., 1996).

The chromosomal localization of the cystatin T gene can be determinedusing methods known in the art. The precise knowledge of a gene'sposition can be useful in a number of ways including: 1) determining ifa sequence is part of an existing contig and obtaining additionalsurrounding genetic sequences in various forms such as YAC-, BAC- orcDNA clones, 2) providing a possible candidate gene for an inheritabledisease which shows linkage to the same chromosomal region, and 3) forcross-referencing model organisms which may be beneficial in helping todetermine what function a particular gene might have.

Known members of the type 2 cystatins are clustered on chromosome 20.Cystatin C (CST3) and D (CST5) are found in the human 20p11 region, andcystatin SN (CST1) and cystatin SA (CST2) are found in the human 20q13.3region. Cystatin T has been mapped to mouse chromosome 2 in the regionsyntenic with the 20p11.2 region of human chromosome 20.

The present invention provides reagents for use in diagnosticapplications. For example, the cystatin T gene, a probe comprisingcystatin T DNA or RNA, or a subsequence thereof can be used to determineif the cystatin T gene is present on a specific chromosome or if amutation has occurred. Detectable chromosomal aberrations at thecystatin T gene locus include, but are not limited to, aneuploidy, genecopy number changes, insertions, deletions, restriction site changes andrearrangements. These aberrations can occur within the coding sequence,within introns, or within flanking sequences, including upstreampromoter and regulatory regions, and may be manifested as physicalalterations within a coding sequence or changes in gene expressionlevel.

In general, these diagnostic methods comprise the steps of (a) obtaininga genetic sample from a patient; (b) incubating the genetic sample witha polynucleotide probe or primer as disclosed above, under conditionswherein the polynucleotide will hybridize to complementarypolynucleotide sequence, to produce a first reaction product; and (iii)comparing the first reaction product to a control reaction product. Adifference between the first reaction product and the control reactionproduct is indicative of a genetic abnormality in the patient. Geneticsamples for use within the present invention include genomic DNA, cDNA,and RNA. The polynucleotide probe or primer can be RNA or DNA, and willcomprise a portion of SEQ ID NO:1, the complement of SEQ ID NO:1, or anRNA equivalent thereof. Suitable assay methods in this regard includemolecular genetic techniques known to those in the art, such asrestriction fragment length polymorphism (RFLP) analysis, short tandemrepeat (STR) analysis employing PCR techniques, ligation chain reaction(Barany, PCR Methods and Applications 1:5-16, 1991), ribonucleaseprotection assays, and other genetic linkage analysis techniques knownin the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian,Chest 108:255-65, 1995). Ribonuclease protection assays (see, e.g.,Ausubel et al., ibid., ch. 4) comprise the hybridization of an RNA probeto a patient RNA sample, after which the reaction product (RNA-RNAhybrid) is exposed to RNase. Hybridized regions of the RNA are protectedfrom digestion. Within PCR assays, a patient's genetic sample isincubated with a pair of polynucleotide primers, and the region betweenthe primers is amplified and recovered. Changes in size or amount ofrecovered product are indicative of mutations in the patient. AnotherPCR-based technique that can be employed is single strand conformationalpolymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications1:34-8, 1991).

Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO:1,other polynucleotide probes, primers, fragments and sequences recitedherein or sequences complementary thereto. Polynucleotide hybridizationis well known in the art and widely used for many applications, see forexample, Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor, N.Y., 1989; Ausubel et al., eds., CurrentProtocols in Molecular Biology, John Wiley and Sons, Inc., N.Y., 1987;Berger and Kimmel, eds., Guide to Molecular Cloning Techniques, Methodsin Enzymology, volume 152, 1987 and Wetmur, Crit. Rev. Biochem. Mol.Biol. 26:227-59, 1990. Polynucleotide hybridization exploits the abilityof single stranded complementary sequences to form a double helixhybrid. Such hybrids include DNA-DNA, RNA-RNA and DNA-RNA.

Hybridization will occur between sequences which contain some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases. Stringent hybridization conditionsencompass temperatures of about 5-25° C. below the thermal melting point(T_(m)) of the hybrid and a hybridization buffer having up to 1 M Na⁺.Higher degrees of stringency at lower temperatures can be achieved withthe addition of formamide which reduces the T_(m) of the hybrid about 1°C. for each 1% formamide in the buffer solution. Generally, suchstringent conditions encompass temperatures of 20-70° C. and ahybridization buffer containing up to 6×SSC and 0-50% formamide. Ahigher degree of stringency can be achieved at temperatures of from40-70° C. with a hybridization buffer having up to 4×SSC and from 0-50%formamide. Highly stringent conditions typically encompass temperaturesof 42-70° C. with a hybridization buffer having up to 1×SSC and 0-50%formamide. Different degrees of stringency can be used duringhybridization and washing to achieve maximum specific binding to thetarget sequence. Typically, the washes following hybridization areperformed at increasing degrees of stringency to remove non-hybridizedpolynucleotide probes from hybridized complexes.

The above conditions are meant to serve as a guide and it is well withinthe abilities of one skilled in the art to adapt these conditions foruse with a particular polypeptide hybrid. The T_(m) for a specifictarget sequence is the temperature (under defined conditions) at which50% of the target sequence will hybridize to a perfectly matched probesequence. Those conditions which influence the T_(m) include, the sizeand base pair content of the polynucleotide probe, the ionic strength ofthe hybridization solution, and the presence of destabilizing agents inthe hybridization solution. Numerous equations for calculating T_(m) areknown in the art, see for example (Sambrook et al., ibid.; Ausubel etal., ibid.; Berger and Kimmel, ibid. and Wetmur, ibid.) and are specificfor DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences ofvarying length. Sequence analysis software such as Oligo 4.0 (publiclyavailable shareware) and Primer Premier (PREMIER Biosoft International,Palo Alto, Calif.) as well as sites on the Internet, are available toolsfor analyzing a given sequence and calculating T_(m) based on userdefined criteria. Such programs can also analyze a given sequence underdefined conditions and suggest suitable probe sequences. Typically,hybridization of longer polynucleotide sequences, >50 bp, is done attemperatures of about 20-25° C. below the calculated T_(m). For smallerprobes, <50 bp, hybridization is typically carried out at the T_(m) or5-10° C. below. This allows for the maximum rate of hybridization forDNA-DNA and DNA-RNA hybrids.

The length of the polynucleotide sequence influences the rate andstability of hybrid formation. Smaller probe sequences, <50 bp, come toequilibrium with complementary sequences rapidly, but may form lessstable hybrids. Incubation times of anywhere from minutes to hours canbe used to achieve hybrid formation. Longer probe sequences come toequilibrium more slowly, but form more stable complexes even at lowertemperatures. Incubations are allowed to proceed overnight or longer.Generally, incubations are carried out for a period equal to three timesthe calculated Cot time. Cot time, the time it takes for thepolynucleotide sequences to reassociate, can be calculated for aparticular sequence by methods known in the art.

The base pair composition of polynucleotide sequence will effect thethermal stability of the hybrid complex, thereby influencing the choiceof hybridization temperature and the ionic strength of the hybridizationbuffer. A-T pairs are less stable than G-C pairs in aqueous solutionscontaining NaCl. Therefore, the higher the G-C content, the more stablethe hybrid. Even distribution of G and C residues within the sequencealso contribute positively to hybrid stability. Base pair compositioncan be manipulated to alter the T_(m) of a given sequence, for example,5-methyldeoxycytidine can be substituted for deoxycytidine and5-bromodeoxuridine can be substituted for thymidine to increase theT_(m). 7-deazo-2′-deoxyguanosine can be substituted for guanosine toreduce dependence on T_(m).

Ionic concentration of the hybridization buffer also effects thestability of the hybrid. Hybridization buffers generally containblocking agents such as Denhardt's solution (Sigma Chemical Co., St.Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO),heparin or SDS, and a Na⁺source, such as SSC (1×SSC: 0.15 M NaCl, 15 mMsodium citrate) or SSPE (1×SSPE: 1.8 M NaCl, 10 mM NaH₂PO₄, 1 mM EDTA,pH 7.7). By decreasing the ionic concentration of the buffer, thestability of the hybrid is increased. Typically, hybridization bufferscontain from between 10 mM-1 M Na⁺. Premixed hybridization solutions arealso available from commercial sources such as Clontech Laboratories(Palo Alto, Calif.) and Promega Corporation (Madison, Wis.) for useaccording to manufacturer's instruction. Addition of destabilizing ordenaturing agents such as formamide, tetralkylammonium salts,guanidinium cations or thiocyanate cations to the hybridization solutionwill alter the T_(m) of a hybrid. Typically, formamide is used at aconcentration of up to 50% to allow incubations to be carried out atmore convenient and lower temperatures. Formamide also acts to reducenon-specific background when using RNA probes.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for isolating DNA and RNA arewell known in the art. It is generally preferred to isolate RNA fromtestis, although DNA can also be prepared using RNA from other tissuesor isolated as genomic DNA. Total RNA can be prepared using guanidineHCl extraction followed by isolation by centrifugation in a CsClgradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)⁺ RNAis prepared from total RNA using the method of Aviv and Leder (Proc.Natl. Acad. Sci. USA 69:1408-1412, 1972). Complementary DNA (cDNA) isprepared from poly(A)⁺RNA using known methods. Polynucleotides encodingcystatin T polypeptides are then identified and isolated by, forexample, hybridization or PCR.

The polynucleotides of the present invention can also be synthesizedusing automated equipment. The current method of choice is thephosphoramidite method. If chemically synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 bp) is technically straightforwardand can be accomplished by synthesizing the complementary strands andthen annealing them. For the production of longer genes (>300 bp),however, special strategies must be invoked, because the couplingefficiency of each cycle during chemical DNA synthesis is seldom 100%.To overcome this problem, synthetic genes (double-stranded) areassembled in modular form from single-stranded fragments that are from20 to 100 nucleotides in length. Gene synthesis methods are well knownin the art. See, for example, Glick and Pasternak, MolecularBiotechnology, Principles & Applications of Recombinant DNA, ASM Press,Washington, D.C., 1994; Itakura et al., Annu. Rev. Biochem. 53: 323-56,1984; and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990.

The present invention further provides counterpart polypeptides andpolynucleotides from other species (orthologs). These species include,but are not limited to, mammalian, avian, amphibian, reptile, fish,insect, and other vertebrate and invertebrate species. Of particularinterest are cystatin T polypeptides from other mammalian species,including human, rat, porcine, ovine, bovine, canine, feline, equine andother primate proteins. Species homologs of the human proteins can becloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, a cDNA can be cloned using mRNA obtained from a tissue or celltype that expresses the protein. Suitable sources of mRNA can beidentified by probing Northern blots with probes designed from thesequences disclosed herein. A library is then prepared from mRNA of apositive tissue of cell line. A cystatin T polypeptide-encoding cDNA canthen be isolated by a variety of methods, such as by probing with acomplete or partial human cDNA or with one or more sets of degenerateprobes based on the disclosed sequences. A cDNA can also be cloned usingthe polymerase chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202),using primers designed from the sequences disclosed herein. Within anadditional method, the cDNA library can be used to transform ortransfect host cells, and expression of the cDNA of interest can bedetected with an antibody to cystatin T polypeptide. Similar techniquescan also be applied to the isolation of genomic clones.

Alternate species polypeptides of cystatin T may have importancetherapeutically. It has been demonstrated that in some cases use of anon-native protein, i.e., protein from a different species, can be morepotent than the native protein. For example, salmon calcitonin has beenshown to be considerably more effective in arresting bone resorptionthan human forms of calcitonin. There are several hypotheses as to whysalmon calcitonin is more potent than human calcitonin in treatment ofosteoporosis. These hypotheses include: 1) salmon calcitonin is moreresistant to degradation; 2) salmon calcitonin has a lower metabolicclearance rate (MCR); and 3) salmon calcitonin may have a slightlydifferent conformation, resulting in a higher affinity for bone receptorsites. Another example is found in the β-endorphin family (Ho et al.,Int. J. Peptide Protein Res. 29:521-24, 1987). Studies have demonstratedthat the peripheral opioid activity of camel, horse, turkey and ostrichβ-endorphins is greater than that of human β-endorphins when isolatedguinea pig ileum was electrostimulated and contractions were measured.Vas deferens from rat, mouse and rabbit were assayed as well. In the ratvas deferens model, camel and horse β-endorphins showed the highestrelative potency. Synthesized rat relaxin was as active as human andporcine relaxin in the mouse symphysis pubis assay (Bullesbach andSchwabe, Eur. J. Biochem. 241:533-7, 1996). Thus, the mouse cystatin Tmolecule of the present invention may have higher potency than the humanendogenous molecule in human cells, tissues and recipients.

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NO:1 and SEQ ID NO:2 represent a single allele of the mousecystatin T gene and polypeptide, and that allelic variation andalternative splicing are expected to occur. Allelic variants can becloned by probing cDNA or genomic libraries from different individualsaccording to standard procedures. Allelic variants of the DNA sequenceshown in SEQ ID NO:1, including those containing silent mutations andthose in which mutations result in amino acid sequence changes, arewithin the scope of the present invention as are proteins which areallelic variants of SEQ ID NO:2. cDNAs generated from alternativelyspliced mRNAs, which retain the properties of the cystatin T polypeptideare included within the scope of the present invention, as arepolypeptides encoded by such cDNAs and mRNAs. Allelic variants andsplice variants of these sequences can be cloned by probing CDNA orgenomic libraries from different individuals or tissues according tostandard procedures known in the art.

The present invention also provides isolated cystatin T polypeptidesthat are substantially homologous to the polypeptides of SEQ ID NO:2 andtheir species orthologs. The term “substantially homologous” is usedherein to denote polypeptides having 50%, preferably 60%, morepreferably at least 80%, sequence identity to the sequences shown in SEQID NO:2 or their orthologs. Such polypeptides will more preferably be atleast 90% identical, and most preferably 95% or more identical to SEQ IDNO:2 or its orthologs. Percent sequence identity is determined byconventional methods. See, for example, Altschul et al., Bull. Math.Bio. 48: 603-66, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci.USA 89:10915-9, 1992. Briefly, two amino acid sequences are aligned tooptimize the alignment scores using a gap opening penalty of 10, a gapextension penalty of 1, and the “blosum 62” scoring matrix of Henikoffand Henikoff (ibid.) as shown in Table 3 (amino acids are indicated bythe standard one-letter codes). The percent identity is then calculatedas:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}} \right. \\{{number}\quad {of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}} \\\left. {{sequence}\quad {in}\quad {order}\quad {to}\quad {align}\quad {the}\quad {two}\quad {sequences}} \right\rbrack\end{matrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Sequence identity of polynucleotide molecules is determined by similarmethods using a ratio as disclosed above.

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant cystatin T. The FASTA algorithm is described by Pearson andLipman, Proc. Nat. Acad. Sci. USA 85:2444, 1988, and by Pearson, Meth.Enzymol. 183:63, 1990.

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:2) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444, 1970); Sellers, SIAM J. Appl. Math. 26:787, 1974), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62. These parameters can be introducedinto a FASTA program by modifying the scoring matrix file (“SMATRIX”),as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63, 1990.

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

The present invention includes nucleic acid molecules that encode apolypeptide having one or more conservative amino acid changes, comparedwith the amino acid sequence of SEQ ID NO:2. The BLOSUM62 table is anamino acid substitution matrix derived from about 2,000 local multiplealignments of protein sequence segments, representing highly conservedregions of more than 500 groups of related proteins (Henikoff andHenikoff, Proc. Nat. Acad. Sci. USA 89:10915, 1992). Accordingly, theBLOSUM62 substitution frequencies can be used to define conservativeamino acid substitutions that may be introduced into the amino acidsequences of the present invention. As used herein, the language“conservative amino acid substitution” refers to a substitutionrepresented by a BLOSUM62 value of greater than −1. For example, anamino acid substitution is conservative if the substitution ischaracterized by a BLOSUM62 value of 0, 1, 2, or 3. Preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).

Substantially homologous proteins and polypeptides are characterized ashaving one or more amino acid substitutions, deletions or additions.These changes are preferably of a minor nature, that is conservativeamino acid substitutions (see Table 4) and other substitutions that donot significantly affect the folding or activity of the protein orpolypeptide; small deletions, typically of one to about 30 amino acids;and small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue, a small linker peptide of up to about20-25 residues, or an affinity tag. Polypeptides comprising affinitytags can further comprise a proteolytic cleavage site between thecystatin T polypeptide and the affinity tag. Preferred such sitesinclude thrombin cleavage sites and factor Xa cleavage sites.

TABLE 4 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline and a-methyl serine) may be substituted for amino acidresidues of cystatin T polypeptides of the present invention. A limitednumber of non-conservative amino acids, amino acids that are not encodedby the genetic code, and unnatural amino acids may be substituted forcystatin T polypeptide amino acid residues. The proteins of the presentinvention can also comprise non-naturally occurring amino acid residues.

Non-naturally occurring amino acids include, without limitation,trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline,trans-4-hydroxyproline, N-methylglycine, allo-threonine,methylthreonine, hydroxy-ethylcysteine, hydroxyethyl-homocysteine,nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenyl-alanine, and4-fluorophenylalanine. Several methods are known in the art forincorporating non-naturally occurring amino acid residues into proteins.For example, an in vitro system can be employed wherein nonsensemutations are suppressed using chemically aminoacylated suppressortRNAs. Methods for synthesizing amino acids and aminoacylating tRNA areknown in the art. Transcription and translation of plasmids containingnonsense mutations is carried out in a cell free system comprising an E.coli S30 extract and commercially available enzymes and other reagents.Proteins are purified by chromatography. See, for example, Robertson etal., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol.202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al.,Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method,translation is carried out in Xenopus oocytes by microinjection ofmutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti etal., J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. colicells are cultured in the absence of a natural amino acid that is to bereplaced (e.g., phenylalanine) and in the presence of the desirednon-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). Thenon-naturally occurring amino acid is incorporated into the protein inplace of its natural counterpart. See, Koide et al., Biochem. 33:7470-6,1994. Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for cystatin T amino acidresidues.

Essential amino acids in the cystatin T polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-5, 1989). Site directedmutagenesis of cystatin C to generate cystatin C variants is discussedin Hall et al., Biochem. J. 291: 123-9, 1993. In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity (e.g., modulating spermatogenesis) to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., J. Biol. Chem. 271:4699-708, 1996. Sites of biologicalinteraction, such as cystatin T polypeptide-cysteine proteinaseinhibitor-enzyme interaction, can also be determined by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., Science 255:306-12,1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al.,FEBS Lett. 309:59-64, 1992. The identities of essential amino acids canalso be inferred from analysis of homologies with related cystatinfamily members.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner etal., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Neret al., DNA 7:127, 1988).

Variants of the disclosed cystatin T DNA and polypeptide sequences canbe generated through DNA shuffling as disclosed by Stemmer, Nature370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

Mutagenesis methods as disclosed above can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides (e.g., cysteine proteaseinhibition or binding) can be recovered from the host cells and rapidlysequenced using modern equipment. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

Polypeptides of the present invention comprise at least 6, preferably atleast 9, more preferably at least 15 contiguous amino acid residues ofSEQ ID NO:2. Within certain embodiments of the invention, thepolypeptides comprise 20, 30, 40, 50 or more contiguous residues of SEQID NO:2, up to the entire predicted mature polypeptide (residues 21-141of SEQ ID NO:2) or the primary translation product (residues 1 to 141 ofSEQ ID NO:2). As disclosed in more detail below, these polypeptides canfurther comprise additional, non-cystatin T, polypeptide sequence(s).Such fragments or peptides may comprise an “immunogenic epitope,” whichis a part of a protein that elicits an antibody response when the entireprotein is used as an immunogen. Immunogenic epitope-bearing peptidescan be identified using standard methods (see, for example, Geysen etal., Proc. Natl. Acad. Sci. USA 81:3998, 1983).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660, 1983).Accordingly, antigenic epitope-bearing peptides and polypeptides of thepresent invention are useful to raise antibodies that bind with thepolypeptides described herein.

Such epitope-bearing peptides and polypeptides can be produced byfragmenting a cystatin T polypeptide, or by chemical peptide synthesis,as described herein. Moreover, epitopes can be selected by phage displayof random peptide libraries (see, for example, Lane and Stephen, Curr.Opin. Immunol. 5:268, 1993), and Cortese et al., Curr. Opin. Biotechnol.7:616, 1996). Standard methods for identifying epitopes and producingantibodies from small peptides that comprise an epitope are described,for example, by Mole, “Epitope Mapping,” in Methods in MolecularBioloqy, Vol. 10, Manson (ed.), pages 105-116 (The Humana Press, Inc.1992), Price, Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages60-84 (Cambridge University Press 1995), and Coligan et al. (eds.),Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages9.4.1-9.4.11 (John Wiley & Sons 1997).

Antibodies that recognize short, linear epitopes are particularly usefulin analytic and diagnostic applications that employ denatured protein,such as Western blotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-6,1979), or in the analysis of fixed cells or tissue samples. Antibodiesto linear epitopes are also useful for detecting fragments of cystatinT, such as might occur in body fluids or cell culture media.

For any cystatin T polypeptide, including variants and fusion proteins,one of ordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 1 and 2 above. Moreover, those of skill in the art canuse standard software to devise cystatin T variants based upon thenucleotide and amino acid sequences described herein. Accordingly, thepresent invention includes a computer-readable medium encoded with adata structure that provides at least one of the following sequences:SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:15. Suitable forms ofcomputer-readable media include magnetic media and optically-readablemedia. Examples of magnetic media include a hard or fixed drive, arandom access memory (RAM) chip, a floppy disk, digital linear tape(DLT), a disk cache, and a ZIP disk. Optically readable media areexemplified by compact discs (e.g., CD-read only memory (ROM),CD-rewritable (RW), and CD-recordable), and digital versatile/videodiscs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).

Using the methods discussed above, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptides that are substantiallyhomologous to residues 21 to 141 of SEQ ID NO:2 or allelic variantsthereof and retain the cysteine protease inhibitory or bindingproperties of the wild-type protein. Such polypeptides may includeadditional amino acids or domains from other members of the cystatinsuperfamily, affinity tags or the like. Cystatin T polypeptide orfragment fusion constructs, containing functional domains of othermembers of the cystatin superfamily, constitute hybrid cysteineproteinase inhibitors exhibiting modified cysteine proteinase inhibitoryor binding capabilities.

Potential single site mutations in the structure of cystatin Tpolypeptides of the present invention can also be identified bystructural analysis. An alignment of cystatin T with murine and humanCRES (SEQ ID NOs:3 and 4), murine and human cystatin C (SEQ ID NOs: 5and 6), human cystatin D (SEQ ID NO:7), human cystatin E (SEQ ID NO:8),human cystatin F (SEQ ID NO:9), human cystatin M (SEQ ID NO:10), humancystatin S (SEQ ID NO:12) and human cystatin SA-I (SEQ ID NO:13) can beused to identify such amino acid residues. Amino acid residues 43 (Val),56 (Asn), 76 Gln), 83 (Tyr), 94 (Cys), 96 (Lys), 104 (Cys), 118 (Cys),127 (Trp) and 138 (Cys) of SEQ ID NO:2 are conserved between thesemembers of the cystatin family. At position 8 of SEQ ID NO:2, mostmembers of the cystatin protein family have either Lys or Ala at thisposition. At position 47 of SEQ ID NO:2, members of the cystatin familyof proteins have either Ala or Cys at that position. At position 51 ofSEQ ID NO:2, members of the cystatin family of proteins have either Alaor Ser at that position. At position 61 of SEQ ID NO:2, members of thecystatin family of proteins have either Asp or Ser at that position. Atposition 63 of SEQ ID NO:2, members of the cystatin family of proteinshave either Tyr or Phe at that position. At position 73 of SEQ ID NO:2,members of the cystatin family of proteins have either Ser or Ala atthat position. At position 87 of SEQ ID NO:2, members of the cystatinfamily of proteins have either Val or Met at that position. At position91 of SEQ ID NO:2, members of the cystatin family of proteins haveeither Arg or Ser at that position. At position 92 of SEQ ID NO:2,members of the cystatin family of proteins have either Thr or Ser atthat position. At position 120 of SEQ ID NO:2, members of the cystatinfamily of proteins have either Phe or Ser at that position. Suggestedmutations for cystatin T polypeptides of the present invention thereforeinclude substitution of the alternate amino acid residue at one or morepositions. Additionally, such substitution could include conservativesubstitutions as shown in Table 4.

Such polypeptides may also include additional polypeptide segments asgenerally disclosed above. For any cystatin T polypeptide, includingvariants and fusion proteins, one of ordinary skill in the art canreadily generate a fully degenerate polynucleotide sequence encodingthat variant using the information set forth in Tables 1 and 2 above.

The polypeptides of the present invention, including full-lengthproteins, fragments thereof and fusion proteins, can be produced ingenetically engineered host cells according to conventional techniques.Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryoticcells, particularly cultured cells of multicellular organisms, arepreferred. Techniques for manipulating cloned DNA molecules andintroducing exogenous DNA into a variety of host cells are disclosed bySambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, andAusubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a cystatin T polypeptide of thepresent invention is operably linked to other genetic elements requiredfor its expression, generally including a transcription promoter andterminator within an expression vector. The vector will also commonlycontain one or more selectable markers and one or more origins ofreplication, although those skilled in the art will recognize thatwithin certain systems selectable markers may be provided on separatevectors, and replication of the exogenous DNA may be provided byintegration into the host cell genome. Selection of promoters,terminators, selectable markers, vectors and other elements is a matterof routine design within the level of ordinary skill in the art. Manysuch elements are described in the literature and are available throughcommercial suppliers.

To direct a cystatin T polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a signal sequence,leader sequence, prepro sequence or pre sequence) is provided in theexpression vector. The secretory signal sequence may be that of thecystatin T polypeptide, or may be derived from another secreted protein(e.g., t-PA) or synthesized de novo. The secretory signal sequence isjoined to the cystatin T polypeptide-encoding DNA sequence in thecorrect reading frame. Secretory signal sequences are commonlypositioned 5′ to the DNA sequence encoding the polypeptide of interest,although certain signal sequences may be positioned elsewhere in the DNAsequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743;Holland et al., U.S. Pat. No. 5,143,830).

Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from amino acid residues1-20 of SEQ ID NO:2 is be operably linked to another polypeptide usingmethods known in the art and disclosed herein. The secretory signalsequence contained in the fusion polypeptides of the present inventionis preferably fused amino-terminally to an additional peptide to directthe additional peptide into the secretory pathway. Such constructs havenumerous applications known in the art. For example, these novelsecretory signal sequence fusion constructs can direct the secretion ofan active component of a normally non-secreted protein, such as areceptor. Such fusions may be used in vivo or in vitro to directpeptides through the secretory pathway. The invention providesconstructs wherein the secretory signal sequence portion of the cystatinT polypeptide (amino acids 1-20 of SEQ ID NO: 2) is employed to directthe secretion of an alternative protein by analogous methods.

Cultured mammalian cells are also suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., eds., Current Protocols in MolecularBiology, John Wiley and Sons, Inc., NY, 1987), liposome-mediatedtransfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al.,Focus 15:80, 1993), and viral vectors (Miller and Rosman, BioTechniques7:980-90, 1989; Wang and Finer, Nature Med. 2:714-6, 1996). Theproduction of recombinant polypeptides in cultured mammalian cells isdisclosed, for example, by Levinson et al., U.S. Pat. No. 4,713,339;Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No.4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable culturedmammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No.CRL 1651), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Grahamet al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g.CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines areknown in the art and available from public depositories such as theAmerican Type Culture Collection, Rockville, Md. In general, strongtranscription promoters are preferred, such as promoters from SV-40 orcytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other suitablepromoters include those from metallothionein genes (U.S. Pat. Nos.4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). DNA encoding the cystatin T polypeptide is inserted into thebaculoviral genome in place of the AcNPV polyhedrin gene coding sequenceby one of two methods. The first is the traditional method of homologousDNA recombination between wild-type AcNPV and a transfer vectorcontaining the cystatin T flanked by AcNPV sequences. Suitable insectcells, e.g. SF9 cells, are infected with wild-type AcNPV and transfectedwith a transfer vector comprising a cystatin T polynucleotide operablylinked to an AcNPV polyhedrin gene promoter, terminator, and flankingsequences. See, King and Possee, The Baculovirus Expression System: ALaboratory Guide, London, Chapman & Hall; O'Reilly et al., BaculovirusExpression Vectors: A Laboratory Manual, New York, Oxford UniversityPress., 1994; and, Richardson (Ed.), Baculovirus Expression Protocols.Methods in Molecular Biology, Totowa, N.J., Humana Press, 1995. Naturalrecombination within an insect cell will result in a recombinantbaculovirus which contains cystatin T driven by the polyhedrin promoter.Recombinant viral stocks are made by methods commonly used in the art.

The second method of making recombinant baculovirus utilizes atransposon-based system described by Luckow et al. (J. Virol.67:4566-79, 1993). This system is sold in the Bac-to-Bac kit (LifeTechnologies, Rockville, Md.). This system utilizes a transfer vector,pFastBac1™ (Life Technologies) containing a Tn7 transposon to move theDNA encoding the cystatin T polypeptide into a baculovirus genomemaintained in E. coli as a large plasmid called a “bacmid.” ThepFastBac1™ transfer vector utilizes the AcNPV polyhedrin promoter todrive the expression of the gene of interest, in this case cystatin T.However, pFastBac1™ can be modified to a considerable degree. Thepolyhedrin promoter can be removed and substituted with the baculovirusbasic protein promoter (also known as Pcor, p6.9 or MP promoter) whichis expressed earlier in the baculovirus infection, and has been shown tobe advantageous for expressing secreted proteins. See, Hill-Perkins andPossee, J. Gen. Virol. 71:971-6, 1990; Bonning et al., J. Gen. Virol.75:1551-6, 1994; and, Chazenbalk and Rapoport, J. Biol. Chem.270:1543-9, 1995. In such transfer vector constructs, a short or longversion of the basic protein promoter can be used. Moreover, transfervectors can be constructed which replace the native cystatin T secretorysignal sequences with secretory signal sequences derived from insectproteins. For example, a secretory signal sequence from EcdysteroidGlucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad,Calif.), or baculovirus gp67 (PharMingen, San Diego, Calif.) can be usedin constructs to replace the native cystatin T secretory signalsequence. In addition, transfer vectors can include an in-frame fusionwith DNA encoding an epitope tag at the C- or N-terminus of theexpressed cystatin T polypeptide, for example, a Glu-Glu epitope tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using atechnique known in the art, a transfer vector containing cystatin T istransformed into E. coli and screened for bacmids which contain aninterrupted lacZ gene indicative of recombinant baculovirus. The bacmidDNA containing the recombinant baculovirus genome is isolated, usingcommon techniques, and used to transfect Spodoptera frugiperda cells,e.g. Sf9 cells. Recombinant virus that expresses cystatin T issubsequently produced. Recombinant viral stocks are made by methodscommonly used the art.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells. Thecells are grown up from an inoculation density of approximately 2-5×10⁵cells to a density of 1-2×10⁶ cells at which time a recombinant viralstock is added at a multiplicity of infection (MOI) of 0.1 to 10, moretypically near 3. The recombinant virus-infected cells typically producethe recombinant cystatin T polypeptide at 12-72 hours post-infection andsecrete it with varying efficiency into the medium. The culture isusually harvested 48 hours post-infection. Centrifugation is used toseparate the cells from the medium (supernatant). The supernatantcontaining the cystatin T polypeptide is filtered through microporefilters, usually 0.45 μm pore size. Procedures used are generallydescribed in available laboratory manuals (King and Possee, ibid.;O'Reilly et al., ibid.; Richardson, ibid.). Subsequent purification ofthe cystatin T polypeptide from the supernatant can be achieved usingmethods described herein.

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein S. cerevisiae is the POT1 vector system disclosed by Kawasaki et al.(U.S. Pat. No. 4,931,373), which allows transformed cells to be selectedby growth in glucose-containing media. Suitable promoters andterminators for use in yeast include those from glycolytic enzyme genes(see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S.Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) and alcoholdehydrogenase genes. See also U.S. Pat. Nos. 4,990,446; 5,063,154;5,139,936 and 4,661,454. Transformation systems for other yeasts,including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyceslactis, Kluyveromyces fragilis, Ustilago maydis, P. pastoris, P.methanolica, P. guillermondii and Candida maltosa are known in the art.See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-65, 1986and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533.

The use of Pichia methanolica as host for the production of recombinantproteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2) Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A preferred selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), whichallows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is preferred to use host cells in which bothmethanol utilization genes (AUG1 and AUG2) are deleted. For productionof secreted proteins, host cells deficient in vacuolar protease genes(PEP4 and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. It is preferred to transform P.methanolica cells by electroporation using an exponentially decaying,pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a cystatin Tpolypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%BaCto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

Expressed recombinant cystatin T polypeptides (or chimeric cystatin Tpolypeptides) can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitableanion exchange media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia,Piscataway, N.J.) being particularly preferred. Exemplarychromatographic media include those media derivatized with phenyl,butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods, Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated byexploitation of their structural features. For example, immobilizedmetal ion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins or proteins having a His-tag. Briefly, a gel isfirst charged with divalent metal ions to form a chelate (E. Sulkowski,Trends in Biochem. 3:1-7, 1985). Histidine-rich proteins will beadsorbed to this matrix with differing affinities, depending upon themetal ion used, and will be eluted by competitive elution, lowering thepH, or use of strong chelating agents. Other methods of purificationinclude purification of glycosylated proteins by lectin affinitychromatography and ion exchange chromatography (Methods in Enzymol.,Vol. 182, “Guide to Protein Purification”, Deutscher, (ed.), Acad.Press, San Diego, 1990, pp.529-39). Within additional embodiments of theinvention, a fusion of the polypeptide of interest and an affinity tag(e.g., polyhistidine, maltose-binding protein, Glu-Glu tag, FLAG tag, animmunoglobulin domain) may be constructed to facilitate purification.

Protein refolding (and optionally reoxidation) procedures may beadvantageously used. It is preferred to purify the protein to >80%purity, more preferably to >90% purity, even more preferably >95%, andparticularly preferred is a pharmaceutically pure state, that is greaterthan 99.9% pure with respect to contaminating macromolecules,particularly other proteins and nucleic acids, and free of infectiousand pyrogenic agents. Preferably, a purified protein is substantiallyfree of other proteins, particularly other proteins of animal origin.

Cystatin T polypeptides or fragments thereof may also be preparedthrough chemical synthesis. Cystatin T polypeptides may be monomers ormultimers; glycosylated or non-glycosylated; pegylated or non-pegylated;and may or may not include an initial methionine amino acid residue.

A cystatin T ligand-binding polypeptide can also be used forpurification of ligand. The polypeptide is immobilized on a solidsupport, such as beads of agarose, cross-linked agarose, glass,cellulosic resins, silica-based resins, polystyrene, cross-linkedpolyacrylamide, or like materials that are stable under the conditionsof use. Methods for linking polypeptides to solid supports are known inthe art, and include amine chemistry, cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, and hydrazide activation. The resulting medium willgenerally be configured in the form of a column, and fluids containingligand are passed through the column one or more times to allow ligandto bind to the receptor polypeptide. The ligand is then eluted usingchanges in salt concentration, chaotropic agents (guanidine HCl), or pHto disrupt ligand-receptor binding.

An assay system that uses a ligand-binding receptor (or an antibody, onemember of a complement/anti-complement pair) or a binding fragmentthereof, and a commercially available biosensor instrument (BIAcore™,Pharmacia Biosensor, Piscataway, N.J.) may be advantageously employed.Such receptor, antibody, member of a complement/anti-complement pair orfragment is immobilized onto the surface of a receptor chip. Use of thisinstrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40,1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. Areceptor, antibody, member or fragment is covalently attached, usingamine or sulfhydryl chemistry, to dextran fibers that are attached togold film within the flow cell. A test sample is passed through thecell. If a ligand, epitope, or opposite member of thecomplement/anti-complement pair is present in the sample, it will bindto the immobilized receptor, antibody or member, respectively, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding. As usedherein, the term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The ability of polypeptides of the present invention to stimulateproliferation or differentiation of testicular cells can be measuredusing cultured testicular cells or in vivo by administering molecules ofthe present invention to the appropriate animal model. Culturedtesticular cells include dolphin DB1.Tes cells (CRL-6258); mouse GC-1spg cells (CRL-2053); TM3 cells (CRL-1714); TM4 cells (CRL-1715); MLTC-1(CRL-2065); I-10 (CCL-83); and pig ST cells (CRL-1746), available fromAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.Assays measuring cell proliferation or differentiation are well known inthe art. For example, assays measuring proliferation include such assaysas chemosensitivity to neutral red dye (Cavanaugh et al.,Investigational New Drugs 8:347-354, 1990), incorporation ofradiolabelled nucleotides (Cook et al., Analytical Biochem. 179:1-7,1989), incorporation of 5-bromo-2′-deoxyuridine (BrdU) in the DNA ofproliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179,1985), and use of tetrazolium salts (Mosmann, J. Immunol. Methods65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall etal., Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res.48:4827-4833, 1988). Assays measuring differentiation include, forexample, measuring cell-surface markers associated with stage-specificexpression of a tissue, enzymatic activity, functional activity ormorphological changes (Watt, FASEB, 5:281-284, 1991; Francis,Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol.Bioprocesses, 161-71, 1989).

In vivo assays for evaluating the effect of polypeptides such ascystatin T on spermatogenesis are well known in the art. For example,compounds can be injected intraperitoneally for a specific timeduration. After the treatment period, animals are sacrificed and testesremoved and weighed. Testicles are homogenized and sperm head counts aremade (Meistrich et al., Exp. Cell Res. 99:72-8, 1976).

Ligand-binding receptor polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-72, 1949) and calorimetric assays (Cunningham et al., Science253:545-8, 1991; Cunningham et al., Science 245:821-5, 1991).

The invention also provides use of cystatin T polypeptides andpolynucleotides in diagnostic applications. Cystatin T polypeptides orpolynucleotides can be used, for example, in assays such as fordetermining circulating levels of polypeptides in a biological sample;or detecting or quantitating soluble polypeptides as marker ofunderlying pathology or disease. Methods for immunochemical detection ofsuch proteins are known, see for example Esnard et al., (FEBS Lett.,1992). Such biological samples include blood, seminal, testis orinterstitial fluids. Cystatin T concentrations can be compared to knowncystatins such as cystatin C or to other testicular proteins such asCRES.

The invention also provides anti-cystatin T antibodies. Antibodies tocystatin T can be obtained, for example, using as an antigen the productof a cystatin T expression vector, or cystatin T isolated from a naturalsource. Particularly useful anti-cystatin T antibodies “bindspecifically” with cystatin T. Antibodies are considered to bespecifically binding if the antibodies bind to a cystatin T polypeptide,peptide or epitope with a binding affinity (K_(a)) of 10⁶ M⁻¹ orgreater, preferably 10⁷ M⁻¹ or greater, more preferably 10⁸ M⁻¹ orgreater, and most preferably 10⁹ M⁻¹ or greater. The binding affinity ofan antibody can be readily determined by one of ordinary skill in theart, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci.51:660, 1949). Suitable antibodies include antibodies that bind withcystatin T in particular domains.

Anti-cystatin T antibodies can be produced using antigenic cystatin Tepitope-bearing peptides and polypeptides. Antigenic epitope-bearingpeptides and polypeptides of the present invention contain a sequence ofat least nine, preferably between 15 to about 30 amino acids containedwithin SEQ ID NO:2. However, peptides or polypeptides comprising alarger portion of an amino acid sequence of the invention, containingfrom 30 to 50 amino acids, or any length up to and including the entireamino acid sequence of a polypeptide of the invention, also are usefulfor inducing antibodies that bind with cystatin T. It is desirable thatthe amino acid sequence of the epitope-bearing peptide is selected toprovide substantial solubility in aqueous solvents (i.e., the sequenceincludes relatively hydrophilic residues, while hydrophobic residues arepreferably avoided). Moreover, amino acid sequences containing prolineresidues may be also be desirable for antibody production.

Polyclonal antibodies to recombinant cystatin T protein or to cystatin Tisolated from natural sources can be prepared using methods well-knownto those of skill in the art. See, for example, Green et al.,“Production of Polyclonal Antisera,” in Immunochemical Protocols(Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995). The immunogenicity of a cystatin T polypeptidecan be increased through the use of an adjuvant, such as alum (aluminumhydroxide) or Freund's complete or incomplete adjuvant. Polypeptidesuseful for immunization also include fusion polypeptides, such asfusions of cystatin T or a portion thereof with an immunoglobulinpolypeptide or with maltose binding protein. The polypeptide immunogenmay be a full-length molecule or a portion thereof. If the polypeptideportion is “hapten-like,” such portion may be advantageously joined orlinked to a macromolecular carrier (such as keyhole limpet hemocyanin(KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such ashorses, cows, dogs, chicken, rats, mice, rabbits, hamsters, guinea pigs,goats, or sheep, an anti-cystatin T antibody of the present inventionmay also be derived from a subhuman primate antibody. General techniquesfor raising diagnostically and therapeutically useful antibodies inbaboons may be found, for example, in Goldenberg et al., internationalpatent publication No. WO 91/11465, and in Losman et al., Int. J. Cancer46:310, 1990. Antibodies can also be raised in transgenic animals suchas transgenic sheep, cows, goats or pigs, and can also be expressed inyeast and fungi in modified forms as will as in mammalian and insectcells.

Alternatively, monoclonal anti-cystatin T antibodies can be generated.Rodent monoclonal antibodies to specific antigens may be obtained bymethods known to those skilled in the art (see, for example, Kohler etal., Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols inImmunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991), Picksleyet al., “Production of monoclonal antibodies against proteins expressedin E. coli,” in DNA Cloning 2: Expression Systems, 2nd Edition, Gloveret al. (eds.), page 93 (Oxford University Press 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a cystatin T gene product, verifying the presenceof antibody production by removing a serum sample, removing the spleento obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to the antigen, culturing the clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

In addition, an anti-cystatin T antibody of the present invention may bederived from a human monoclonal antibody. Human monoclonal antibodiesare obtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described, for example, by Green et al., NatureGenet. 7:13, 1994, Lonberg et al., Nature 368:856, 1994, and Taylor etal., Int. Immun. 6:579, 1994.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments ofanti-cystatin T antibodies. Such antibody fragments can be obtained, forexample, by proteolytic hydrolysis of the antibody. Antibody fragmentscan be obtained by pepsin or papain digestion of whole antibodies byconventional methods. As an illustration, antibody fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent to produce 3.5S Fab′ monovalent fragments.Optionally, the cleavage reaction can be performed using a blockinggroup for the sulfhydryl groups that result from cleavage of disulfidelinkages. As an alternative, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. No. 4,331,647,Nisonoff et al., Arch Biochem. Biophys. 89:230, 1960, Porter, Biochem.J. 73:119, 1959, Edelman et al., in Methods in Enzymology Vol. 1, page422 (Academic Press 1967), and by Coligan, ibid.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described by Inbar etal., Proc. Natl. Acad. Sci. USA 69:2659, 1972. Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as gluteraldehyde (see, for example,Sandhu, Crit. Rev. Biotech. 12:437, 1992).

The Fv fragments may comprise V_(H) and V_(L) chains which are connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains which are connected by anoligonucleotide. The structural gene is inserted into an expressionvector which is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are described, for example, by Whitlow et al., Methods: ACompanion to Methods in Enzymology 2:97, 1991, also see, Bird et al.,Science 242:423, 1988, Ladner et al., U.S. Pat. No. 4,946,778, Pack etal., Bio/Technology 11:1271, 1993, and Sandhu, supra.

As an illustration, a scFV can be obtained by exposing lymphocytes tocystatin T polypeptide in vitro, and selecting antibody displaylibraries in phage or similar vectors (for instance, through use ofimmobilized or labeled cystatin T protein or peptide). Genes encodingpolypeptides having potential cystatin T polypeptide binding domains canbe obtained by screening random peptide libraries displayed on phage(phage display) or on bacteria, such as E. coli. Nucleotide sequencesencoding the polypeptides can be obtained in a number of ways, such asthrough random mutagenesis and random polynucleotide synthesis. Theserandom peptide display libraries can be used to screen for peptideswhich interact with a known target which can be a protein orpolypeptide, such as a ligand or receptor, a biological or syntheticmacromolecule, or organic or inorganic substances. Techniques forcreating and screening such random peptide display libraries are knownin the art (Ladner et al., U.S. Pat. No. 5,223,409, Ladner et al., U.S.Pat. No. 4,946,778, Ladner et al., U.S. Pat. No. 5,403,484, Ladner etal., U.S. Pat. No. 5,571,698, and Kay et al., Phage Display of Peptidesand Proteins (Academic Press, Inc. 1996)) and random peptide displaylibraries and kits for screening such libraries are availablecommercially, for instance from Clontech (Palo Alto, Calif.), InvitrogenInc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.),and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Random peptidedisplay libraries can be screened using the cystatin T sequencesdisclosed herein to identify proteins which bind to cystatin T.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106, 1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an anti-cystatin T antibody may be derived from a“humanized” monoclonal antibody. Humanized monoclonal antibodies areproduced by transferring mouse complementary determining regions fromheavy and light variable chains of the mouse immunoglobulin into a humanvariable domain. Typical residues of human antibodies are thensubstituted in the framework regions of the murine counterparts. The useof antibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions. General techniques for cloning murine immunoglobulinvariable domains are described, for example, by Orlandi et al., Proc.Nat. Acad. Sci. USA 86:3833, 1989. Techniques for producing humanizedmonoclonal antibodies are described, for example, by Jones et al.,Nature 321:522, 1986, Carter et al., Proc. Nat. Acad. Sci. USA 89:4285,1992, Sandhu, Crit. Rev. Biotech. 12:437, 1992, Singer et al., J. Immun.150:2844, 1993, Sudhir (ed.), Antibody Engineering Protocols (HumanaPress, Inc. 1995), Kelley, “Engineering Therapeutic Antibodies,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S.Pat. No. 5,693,762 (1997).

Polyclonal anti-idiotype antibodies can be prepared by immunizinganimals with anti-cystatin T antibodies or antibody fragments, usingstandard techniques. See, for example, Green et al., “Production ofPolyclonal Antisera,” in Methods In Molecular Biology: ImmunochemicalProtocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, seeColigan, ibid. at pages 2.4.1-2.4.7. Alternatively, monoclonalanti-idiotype antibodies can be prepared using anti-cystatin Tantibodies or antibody fragments as immunogens with the techniques,described above. As another alternative, humanized anti-idiotypeantibodies or subhuman primate anti-idiotype antibodies can be preparedusing the above-described techniques. Methods for producinganti-idiotype antibodies are described, for example, by Irie, U.S. Pat.No. 5,208,146, Greene, et. al., U.S. Pat. No. 5,637,677, and Varthakaviand Minocha, J. Gen. Virol. 77:1875, 1996.

Genes encoding polypeptides having potential cystatin T polypeptidebinding domains, “binding proteins”, can be obtained by screening randomor directed peptide libraries displayed on phage (phage display) or onbacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. Alternatively,constrained phage display libraries can also be produced. These peptidedisplay libraries can be used to screen for peptides which interact witha known target which can be a protein or polypeptide, such as a ligandor receptor, a biological or synthetic macromolecule, or organic orinorganic substances. Techniques for creating and screening such peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andpeptide display libraries and kits for screening such libraries areavailable commercially, for instance from Clontech (Palo Alto, Calif.),Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly,Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Peptidedisplay libraries can be screened using the cystatin T sequencesdisclosed herein to identify proteins which bind to cystatin T. These“binding proteins” which interact with cystatin T polypeptides can beused essentially like an antibody.

A variety of assays known to those skilled in the art can be utilized todetect antibodies and/or binding proteins which specifically bind tocystatin T proteins or peptides. Exemplary assays are described indetail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), ColdSpring Harbor Laboratory Press, 1988. Representative examples of suchassays include: concurrent immunoelectrophoresis, radioimmunoassay,radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dotblot or Western blot assay, inhibition or competition assay, andsandwich assay. In addition, antibodies can be screened for binding towild-type versus mutant cystatin T protein or polypeptide.

Antibodies and binding proteins to cystatin T may be used for taggingcells that express cystatin T; for isolating cystatin T by affinitypurification; for diagnostic assays for determining circulating levelsof cystatin T polypeptides; for detecting or quantitating solublecystatin T as marker of underlying pathology or disease; in analyticalmethods employing FACS; for screening expression libraries; forgenerating anti-idiotypic antibodies; and as neutralizing antibodies oras antagonists to block cystatin T polypeptide modulation ofspermatogenesis or like activity in vitro and in vivo. Suitable directtags or labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent markers, chemiluminescent markers, magneticparticles and the like; indirect tags or labels may feature use ofbiotin-avidin or other complement/anti-complement pairs asintermediates. Moreover, antibodies to cystatin T or fragments thereofmay be used in vitro to detect denatured cystatin T or fragments thereofin assays, for example, Western Blots or other assays known in the art.

Antibodies or polypeptides herein can also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications. Forinstance, polypeptides or antibodies of the present invention can beused to identify or treat tissues or organs that express a correspondinganti-complementary molecule (receptor or antigen, respectively, forinstance). More specifically, cystatin T polypeptides or anti-cystatin Tantibodies, or bioactive fragments or portions thereof, can be coupledto detectable or cytotoxic molecules and delivered to a mammal havingcells, tissues or organs that express the anti-complementary molecule.

Molecules of the present invention can be used to identify and isolatecysteine proteinases with which cystatin T polypeptide interacts. Forexample, proteins and peptides of the present invention can beimmobilized on a column and membrane preparations run over the column(Immobilized Affinity Ligand Techniques, Hermanson et al., eds.,Academic Press, San Diego, Calif., 1992, pp.195-202). Proteins andpeptides can also be radiolabeled (Methods in Enzymol., vol. 182, “Guideto Protein Purification”, M. Deutscher, ed., Acad. Press, San Diego,1990, 721-37) or photoaffinity labeled (Brunner et al., Ann. Rev.Biochem. 62:483-514, 1993 and Fedan et al., Biochem. Pharmacol.33:1167-80, 1984) and specific cysteine proteinases can be identified.

Assays known in the art for evaluating cysteine protease inhibition maybe employed to identify or evaluate cystatin T polypeptide agonists,antagonists, homologs, paralogs, and the like. Such assays include thosedescribed by Sotiropoulou et al., J. Biol. Chem. 272: 903-10, 1997(papain assay); Adenis et al., Cancer Letters 96: 267-75, 1995(cathepsin B, L and D activity assays); Hall et al., Biochem. J. 291:123-9, 1993 (enzyme inhibition assays); Laszlo et al., Acta PaediatricaHungarica 28: 175-78, 1987 (activity of cathepsins B, H and L in theserum of cystic fibrosis patients); Luthgens et al., Cancer Detectionand Prevention 17: 387-97, 1993 (bronchoalveolar lavage methods);Luisetti et al., Respiration 59: 24-7, 1992 (bronchoalveolar lavageevaluation of protease-anti-protease imbalance); and the like.

Assays known in the art for evaluating urokinase-type plasminogenactivator (uPA) may also be used to identify or evaluate cystatin Tpolypeptide agonists, antagonists, homologs, paralogs, and the like.Such assays include those described by Silberman et al., J. Biol. Chem.272: 5927-35, 1997 (Northern analysis); Nauland & Rijken, Eur. J.Biochem. 223: 497-501, 1994 (two chain uPA activity); Schmitt et al.,Biol. Chem. 373: 611-22, 1992 (quantitative assessment of uPA andproteolytic factors in tumor tissue extracts); Kobayashi et al., J.Biol. Chem. 266: 5147-52, 1991 (assays for enzymatic activity andPro-uPA-cathepsin B or D interaction); and the like.

An additional aspect of the present invention provides methods foridentifying agonists or antagonists of the cystatin T polypeptidesdisclosed above, which agonists or antagonists may have valuabletherapeutic properties as discussed further herein. Within oneembodiment, there is provided a method of identifying cystatin Tpolypeptide agonists, comprising providing cells responsive to acystatin T polypeptide, culturing the cells in the presence of a testcompound and comparing the cellular response with the cell cultured inthe presence of the cystatin T polypeptide, and selecting the testcompounds for which the cellular response is of the same type.Alternatively, putative agonists of cystatin T polypeptide can beevaluated as generally described above using cysteine proteinase bindingassays. If the putative agonist binds cysteine proteinases, such as theplant proteinase papain, with an affinity within an order of magnitudebelow the cystatin T polypeptide or higher, that putative agonist isselected as an agonist.

Within another embodiment, there is provided a method of identifyingantagonists of cystatin T polypeptide, comprising providing cellsresponsive to a cystatin T polypeptide, culturing a first portion of thecells in the presence of cystatin T polypeptide, culturing a secondportion of the cells in the presence of the cystatin T polypeptide and atest compound, and detecting a decrease in a cellular response of thesecond portion of the cells as compared to the first portion of thecells. Alternatively, putative antagonists of cystatin T polypeptide canbe evaluated as generally described above using cysteine proteinasebinding assays. If the putative antagonist inhibits cystatin Tpolypeptide binding to cysteine proteinases, such as the plantproteinase papain, by at least one order of magnitude, that putativeantagonist is selected as an antagonist.

Cystatin T polypeptide agonists are useful in applications requiringmodulation of spermatogenesis, such as in in vitro or in vivo study ofsperm development and maturation. Cystatin T polypeptide antagonists areuseful in applications requiring inhibition of spermatogenesis, such asin in vitro or in vivo study of fertilization and conception.

Polynucleotides encoding cystatin T polypeptides are useful within genetherapy applications where it is desired to increase or inhibit cystatinT activity. If a mammal has a mutated or absent cystatin T gene, thecystatin T gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a cystatin T polypeptide is introduced invivo in a viral vector. Such vectors include an attenuated or defectiveDNA virus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-30, 1991); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30,1992; and a defective adeno-associated virus vector (Samulski et al., J.Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

In another embodiment, a cystatin T gene can be introduced in aretroviral vector, e.g., as described in Anderson et al., U.S. Pat. No.5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263;International Patent Publication NO: WO 95/07358, published Mar. 16,1995 by Dougherty et al.; and Kuo et al., Blood 82:845, 1993.Alternatively, the vector can be introduced by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl.Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. More particularly, directingtransfection to particular cells represents one area of benefit. Forinstance, directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, suchas the pancreas, liver, kidney, and brain. Lipids may be chemicallycoupled to other molecules for the purpose of targeting. Targetedpeptides (e.g., hormones or neurotransmitters), proteins such asantibodies, or non-peptide molecules can be coupled to liposomeschemically.

It is possible to remove the target cells from the body; to introducethe vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

Antisense methodology can be used to inhibit cystatin T genetranscription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of a cystatinT-encoding polynucleotide (e.g., a polynucleotide as set froth in SEQ IDNO:1) are designed to bind to cystatin T-encoding mRNA and to inhibittranslation of such mRNA. Such antisense polynucleotides are used toinhibit expression of cystatin T polypeptide-encoding genes in cellculture or in a subject.

Transgenic mice, engineered to express the cystatin T gene, and micethat exhibit a complete absence of cystatin T gene function, referred toas “knockout mice” (Snouwaert et al., Science 257:1083, 1992), may alsobe generated (Lowell et al., Nature 366:740-2, 1993). These mice may beemployed to study the cystatin T gene and the protein encoded thereby inan in vivo system.

For pharmaceutical use, the proteins of the present invention areformulated for parenteral, such as intravenous or subcutaneous, deliveryaccording to conventional methods. Other modes of administration includetablets, caplets, pills, powders, granules, eyedrops, oral or ocularsolutions or suspensions, ocular ointments, transdermal patches andoil-in-water emulsions. Intravenous administration will be by bolusinjection or infusion over a typical period of one to several hours. Ingeneral, pharmaceutical formulations will include a cystatin T proteinin combination with a pharmaceutically acceptable vehicle, such assaline, buffered saline, 5% dextrose in water or the like. Formulationsmay further include one or more excipients, preservatives, solubilizers,buffering agents, albumin to prevent protein loss on vial surfaces, etc.Methods of formulation are well known in the art and are disclosed, forexample, in Remington: The Science and Practice of Pharmacy, Gennaro,ed., Mack Publishing Co., Easton, Pa., 19th ed., 1995.

As used herein a “pharmaceutically effective amount” of a cystatin Tpolypeptide, agonist or antagonist is an amount sufficient to induce adesired biological result. The result can be alleviation of the signs,symptoms, or causes of a disease, or any other desired alteration of abiological system. For example, an effective amount of a cystatin Tpolypeptide is that which provides either subjective relief of symptomsor an objectively identifiable improvement as noted by the clinician orother qualified observer. Effective amounts of the cystatin Tpolypeptides can vary widely depending on the disease or symptom to betreated. The amount of the polypeptide to be administered and itsconcentration in the formulations, depends upon the vehicle selected,route of administration, the potency of the particular polypeptide, theclinical condition of the patient, the side effects and the stability ofthe compound in the formulation. Thus, the clinician will employ theappropriate preparation containing the appropriate concentration in theformulation, as well as the amount of formulation administered,depending upon clinical experience with the patient in question or withsimilar patients. Such amounts will depend, in part, on the particularcondition to be treated, age, weight, and general health of the patient,and other factors evident to those skilled in the art. Doses forspecific compounds may be determined from in vitro or ex vivo studies incombination with studies on experimental animals.

The dosages of the present compounds used to practice the inventioninclude dosages effective to result in the desired effects. Estimationof appropriate dosages effective for the individual patient is wellwithin the skill of the ordinary prescribing physician or otherappropriate health care practitioner. As a guide, the clinician can useconventionally available advice from a source such as the Physician'sDesk Reference, 48^(th) Edition, Medical Economics Data Production Co.,Montvale, N.J. 07645-1742 (1994).

The present invention, thus generally described, will be understood morereadily by reference to the following example, which is provided by wayof illustration and is not intended to be limiting of the presentinvention.

EXAMPLES Example 1 Identification of the Cystatin T Sequence

The novel cystatin T polypeptide-encoding polynucleotides of the presentinvention were initially identified by querying an EST database forcystatin homologs. An EST discovered and predicted to be related to thecystatin family, but lacked the 5′ half the polynucleotide sequence.Oligonucleotides ZC17516 (SEQ ID NO:21) and ZC17517 (SEQ ID NO:22)derived from the EST sequence were used as primers to amplify the regionfrom a variety of cDNA libraries. Amplification of the sequence occurredonly when using testis libraries. To identify the corresponding fulllength cDNA, 5′ RACE was used to obtain the missing sequence. Murinetestis cDNA was used as a template and oligonucleotides ZC18696 (SEQ IDNO:16) and ZC18369 (SEQ ID NO:17) were used a primers. The sequence ofthe cloned PCR produce was confirmed by sequence analysis. Using anInvitrogen S.N.A.P.™ Miniprep kit (Invitrogen, Corp., San Diego, Calif.)according to manufacturer's instructions a 5 ml overnight culture inLB+50 μg/ml ampicillin was prepared. The template was sequenced on anABIPRISM™ model 377 DNA sequencer (Perkin-Elmer Cetus, Norwalk, Conn.)using the ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit(Perkin-Elmer Corp.) according to manufacturer's instructions.Sequencing reactions were carried out in a Hybaid OmniGene TemperatureCycling System (National Labnet Co., Woodbridge, N.Y.). SEQUENCHER™ 3.0sequence analysis software (Gene Codes Corporation, Ann Arbor, Mich.)was used for data analysis. The resulting 490 bp sequence murinecystatin T sequence is disclosed in SEQ ID NO:1.

Example 2 Tissue Distribution

Mouse Multiple Tissue Northern Blot (Clontech) was probed to determinethe murine tissue distribution of murine cystatin T expression. An 188bp DNA probe derived from the clone described above that contains the 3′end of the cystatin T polynucleotide sequence (SEQ ID NO:1) including 30bp 3′UTR was generated by PCR and mouse testis cDNA as a template. Theprobe was radioactively using a Rediprime II DNA Labeling System(Amersham Pharmacia Biotech, Inc., Piscataway, N.J.) according to themanufacturer's specifications. The probe was purified using a NUCTRAPpush column (Stratagene Cloning Systems, La Jolla, Calif.). ExpressHyb™(Clontech) solution was used for prehybridization and as a hybridizingsolution for the Northern blots. Hybridization took place overnight at65° C. using 1.0×10⁶ cpm/ml of labeled probe, and the blots were thenwashed at 4 times at 25° C. in 2×SSC, 0.1% SDS, followed by 2 washes at50° C. in 0.1×SSC, 0.1% SDS and one wash at 56° C. in 0.1×SSC, 0.1% SDS.A 1.0 kb transcript was detected in testis tissue only.

A mouse multiple tissue sub-blot was prepared by flash freezing mousebladder, colon, epididymus, prostate, seminal vesicle, testis, wasdeferens, ovary and uterus tissues in liquid nitrogen. The tissues werethen processed using RNeasy® Total RNA System (Qiagen) according tomanufacturer's instruction. Twenty milligrams of the total RNA from eachtissue sample separated by 1.5% agarose mini gel (Stratagene CloningSystems, La Jolla, Calif.) electrophoresis in formaldehyde/phosphatebuffer. The RNA was blotted overnight onto a nytran filter (Schleicher &Schuell, Keene, N.H.) and the filter was UV crosslinked (1,200 μJoules)in a STRATALINKER® 2400 UV crosslinker (Stratagene Cloning Systems) andthen baked at 80° C. for 30 minutes. Hybridization was as describedabove. Expression was detected in testis only.

A mouse RNA Master Blot™ (Clontech) was probed as described above.Expression was detected only in testis.

A mouse Embryo Northern Blot (Clontech) was also probed to determineexpression of cystatin T in embryonic mouse tissue. Northern blotanalysis was performed as described above. A 1.0 kb transcript wasdetected in 7 day old tissue only.

An interspecies Zoo blot (Clontech) was probed as described above. Theblots were then washed 4 times at 25° C. in 2×SSC, 0.1% SDS, followed by3 washes at 50° C. in 0.1×SSC, 0.1% SDS. A single band is present inmouse and rabbit, two bands are present in rat and potentially in humanand monkey.

A mouse testis blot was probed as described above. The blot was preparedfrom TM3, a murine Leydig cell line (ATCC No: CRL-1714), a murineSertoli cell line TM4 (ATCC No: CRL-1715), two murine Leydig tumor celllines, MLTC-1 (ATCC No: CRL-2065) and I-10 (ATCC No:CCL-83) and a germcell line, GC-1 spg (ATCC No:CRL-2053). Total cytoplasmic RNA wasisolated essentially as described by Davis et al., Preparation andanalysis of RNA from eukaryotic cell. In Basic Methods in MolecularBiology, Elsevier Science Publishing Co., Inc. New York, pp 130-5, 1986.Mouse testis and liver tissue were homogenized using the TH115 TissueHomogenizer (Omni International, Warrenton, Va.). Total RNA was isolatedusing the RNeasy® Total RNA System (Qiagen) according to manufacturer'sinstruction. Twenty milligrams of the total RNA from each tissue sampleseparated by 1% agarose mini gel (Stratagene Cloning Systems, La Jolla,Calif.) electrophoresis in formaldehyde/phosphate buffer. The RNA wasblotted overnight onto a GeneScreen membrane (NEN, Boston, Mass.) andthe filter was UV crosslinked (1,200 μJoules) in a STRATALINKER® 2400 UVcrosslinker (Stratagene Cloning Systems) and then baked at 80° C. for 30minutes. Hybridization was as described above. Expression was detectedin testis only.

Rat organ blots were generated using brain, heart, kidney, liver, lung,pancreas, skeletal muscle, spleen and testis tissue from Sprague Dawleyrats (67 days old) and epididymis, prostate, testis, ovary, uterinetissue from 90 day old Sprague Dawley rats. Probing with ³²P labeledfull length murine Cystatin T indicated that expression was only intestis.

Example 3 Chromosomal Localization

Murine cystatin T was mapped by PCR using commercially available mouseT31 whole genome radiation hybrid (WGRH) panel (Research Genetics, Inc.,Huntsville, Ala.) and Map Manager QT linkage analysis program. The T 31WGRH panel contains DNA from each of 100 radiation hybrid clones, plustwo control DNAs (the 129aa donor and the A23 recipient). For themapping of murine cystatin T with the T31 WGRH panel, 20 μl reactionswere set up in 96-well microtiter plates (Stratagene Cloning Systems, LaJolla, Calif.) and used in RoboCycler Gradient 96 thermal cyclers(Strategen). Each of the 102 PCR reactions consisted of 2 μl 10×KlenTaqPCR reaction buffer (Clontech, Palo Alto, Calif.), 1.6 μl dNTPs mix (2.5mM each, Perkin-Elmer Cetus, Norwalk, Conn.), 1 μl sense primer ZC 20814(SEQ ID NO:19), 1 μl antisense primer ZC20815 (SEQ ID NO:20), 2 μlRediLoad (Research Genetics, Inc.), 0.4 μl 50×Advantage KlenTaqPolymerase Mix (Clontech), 25 ng of DNA from an individual hybrid cloneor control and ddH₂O for a total volume of 20 μl. The PCR cycleconditions were as follows: 5 minutes at 94° C. (1 cycle), followed by45 seconds at 94° C., 45 seconds at 62° C. and 1 minute and 15 secondsat 72° C. (35 cycles) followed by a final extension of 7 minutes at 72°C. The reactions were separated by electrophoresis on a 2% agarose gel(Life Technologies, Gaithersburg, Md.).

At P=0.0001, murine zcys3 linked to the marker D2Mit194 with a LOD scoreof 9.07. D2Mit194 has been mapped at 81.4 cM on mouse chromosome 2. Thisis a known region of synteny or linkage conservation with the 20p11.2region of human chromosome 20, where a cystatin gene cluster has beenmapped containing CST1-5 and 2 pseudogenes.

Example 4 Stage Specific Northern Analysis

To determine if the gene encoding murine Cystatin T is regulated duringthe cycle of the seminiferous epithelium, stage synchronized testes wereobtained from adult Sprague-Dawley rats as described by Wright et al.(Biology of Reproduction 29:257-70, 1983). Seminiferous tubule fragmentscorresponding to the following stages, I, II-III, IV-V, VI, VIIab,VIIcd, VIII, IX-XI, XII, XIII-XIV, in the cycle of the seminiferousepithelium were used. The isolated tubule fragments were solublized inTriZol (Life Technologies) and total RNA was isolated according tomanufacturer's instructions. The total RNA was quantitated and 7.5 μg oftotal RNA from each stage was fractionated using a 1.2% agarose gel in1% formaldehyde and MOPS buffer, transferred to nylon membrane, andcross-linked. The blots were probed with ³²P labeled, full length murineCystatin T DNA as described above. The probe was heat denatured andadded to prehybridization solution for overnight hybridization. Blotswere washed at room temperature, 2×SSC/0.1% SDS for 10 minutes, the 65°C. 0.2% SSC/0.1% SDS, twice for 15 minutes, then exposed to film. As aloading control, the blots were probed with ³²P-labeled CHO B, alsoknown as ribosomal protein S2 (Tohgo et al., FEBS Lett. 340:133-8,1994).

The results of the Northern Blot analysis indicate that the murineCystatin T gene is regulated during the cycle of the seminiferousepithelium. Table 5 shows the amount of the Cystatin T gene relative toCHO B at the various stages tested.

TABLE 5 Fragment Cystatin T/CHO B I 0.04 II-III 0.00 IV-V 0.0 VI 0.01VIIab 0.252 VIIcd 0.670 VIII 1.0 IX-XI 0.954 IX-XIII 0.825 XIV 0.0

The gene encoding murine Cystatin T is highly regulated during the cycleof the seminiferous epithelium. It is very low or undetectable duringstages XIV-VI and rises dramatically during stages VIIab and XII, thendecreased abruptly.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

22 1 490 DNA Mus musculus CDS (46)...(468) 1 gaaagaaaat aggaacttggtatgttcctt gaatgaagaa gcacc atg gcc aga ttc 57 Met Ala Arg Phe 1 tta cagacc ctg ctg ttc ctg gtg atc acg gtg gag ttt gta tct aga 105 Leu Gln ThrLeu Leu Phe Leu Val Ile Thr Val Glu Phe Val Ser Arg 5 10 15 20 aga gtcgaa gcc tgg ggc tcc cca cag att gtg agg cca ttc gaa gac 153 Arg Val GluAla Trp Gly Ser Pro Gln Ile Val Arg Pro Phe Glu Asp 25 30 35 atc ccc aaatcc tat gtc tat gtg cag cat gca ctc tgg tat gcc atg 201 Ile Pro Lys SerTyr Val Tyr Val Gln His Ala Leu Trp Tyr Ala Met 40 45 50 aaa gaa tat aacaag gcc agc aat gac ctc tac aac ttc agg gtg gtg 249 Lys Glu Tyr Asn LysAla Ser Asn Asp Leu Tyr Asn Phe Arg Val Val 55 60 65 gat atc cta aaa tctcag gag cag atc aca gac agt ctg gag tat tat 297 Asp Ile Leu Lys Ser GlnGlu Gln Ile Thr Asp Ser Leu Glu Tyr Tyr 70 75 80 ctt gaa gta aac att gcccga aca atg tgc aag aag att gca gga gat 345 Leu Glu Val Asn Ile Ala ArgThr Met Cys Lys Lys Ile Ala Gly Asp 85 90 95 100 aat gaa aac tgc ttg tttcaa cag gat cct aaa atg aaa aag atg gtg 393 Asn Glu Asn Cys Leu Phe GlnGln Asp Pro Lys Met Lys Lys Met Val 105 110 115 ttt tgc att ttt att gttagc tcc aaa cca tgg aag ttt gaa ctt aaa 441 Phe Cys Ile Phe Ile Val SerSer Lys Pro Trp Lys Phe Glu Leu Lys 120 125 130 atg ctg aag aag caa tgcaaa gat atc taatcagcat tcgggacacc 488 Met Leu Lys Lys Gln Cys Lys AspIle 135 140 tt 490 2 141 PRT Homo sapiens 2 Met Ala Arg Phe Leu Gln ThrLeu Leu Phe Leu Val Ile Thr Val Glu 1 5 10 15 Phe Val Ser Arg Arg ValGlu Ala Trp Gly Ser Pro Gln Ile Val Arg 20 25 30 Pro Phe Glu Asp Ile ProLys Ser Tyr Val Tyr Val Gln His Ala Leu 35 40 45 Trp Tyr Ala Met Lys GluTyr Asn Lys Ala Ser Asn Asp Leu Tyr Asn 50 55 60 Phe Arg Val Val Asp IleLeu Lys Ser Gln Glu Gln Ile Thr Asp Ser 65 70 75 80 Leu Glu Tyr Tyr LeuGlu Val Asn Ile Ala Arg Thr Met Cys Lys Lys 85 90 95 Ile Ala Gly Asp AsnGlu Asn Cys Leu Phe Gln Gln Asp Pro Lys Met 100 105 110 Lys Lys Met ValPhe Cys Ile Phe Ile Val Ser Ser Lys Pro Trp Lys 115 120 125 Phe Glu LeuLys Met Leu Lys Lys Gln Cys Lys Asp Ile 130 135 140 3 142 PRT Musmusculus 3 Met Ala Lys Pro Leu Trp Leu Ser Leu Ile Leu Phe Ile Ile ProVal 1 5 10 15 Ala Leu Ala Val Gly Val Asp Gln Ser Lys Asn Glu Val LysAla Gln 20 25 30 Asn Tyr Phe Gly Ser Ile Asn Ile Ser Asn Ala Asn Val LysGln Cys 35 40 45 Val Trp Phe Ala Met Lys Glu Tyr Asn Lys Glu Ser Glu AspLys Tyr 50 55 60 Val Phe Leu Val Asp Lys Ile Leu His Ala Lys Leu Gln IleThr Asp 65 70 75 80 Arg Met Glu Tyr Gln Ile Asp Val Gln Ile Ser Arg SerAsn Cys Lys 85 90 95 Lys Pro Leu Asn Asn Thr Glu Asn Cys Ile Pro Gln LysLys Pro Glu 100 105 110 Leu Glu Lys Lys Met Ser Cys Ser Phe Leu Val GlyAla Leu Pro Trp 115 120 125 Asn Gly Glu Phe Asn Leu Leu Ser Lys Glu CysLys Asp Val 130 135 140 4 142 PRT Homo sapiens 4 Met Pro Arg Cys Arg TrpLeu Ser Leu Ile Leu Leu Thr Ile Pro Leu 1 5 10 15 Ala Leu Val Ala ArgLys Asp Pro Lys Lys Asn Glu Thr Gly Val Leu 20 25 30 Arg Lys Leu Lys ProVal Asn Ala Ser Asn Ala Asn Val Lys Gln Cys 35 40 45 Leu Trp Phe Ala MetGln Glu Tyr Asn Lys Glu Ser Glu Asp Lys Tyr 50 55 60 Val Phe Leu Val ValLys Thr Leu Gln Ala Gln Leu Gln Val Thr Asn 65 70 75 80 Leu Leu Glu TyrLeu Ile Asp Val Glu Ile Ala Arg Ser Asp Cys Arg 85 90 95 Lys Pro Leu SerThr Asn Glu Ile Cys Ala Ile Gln Glu Asn Ser Lys 100 105 110 Leu Lys ArgLys Leu Ser Cys Ser Phe Leu Val Gly Ala Leu Pro Trp 115 120 125 Asn GlyGlu Phe Thr Val Met Glu Lys Lys Cys Glu Asp Ala 130 135 140 5 140 PRTMus musculus 5 Met Ala Ser Pro Leu Arg Ser Leu Leu Phe Leu Leu Ala ValLeu Gly 1 5 10 15 Val Ala Trp Ala Ala Thr Pro Lys Gln Gly Pro Arg MetLeu Gly Ala 20 25 30 Pro Glu Glu Ala Asp Ala Asn Glu Glu Gly Val Arg ArgAla Leu Asp 35 40 45 Phe Ala Val Ser Glu Tyr Asn Lys Gly Ser Asn Asp AlaTyr His Ser 50 55 60 Arg Ala Ile Gln Val Val Arg Ala Arg Lys Gln Leu ValAla Gly Val 65 70 75 80 Asn Tyr Phe Phe Asp Val Glu Met Gly Arg Thr ThrCys Thr Lys Ser 85 90 95 Gln Thr Asn Leu Thr Asp Cys Pro Phe His Asp GlnPro His Leu Met 100 105 110 Arg Lys Ala Leu Cys Ser Phe Gln Ile Tyr SerVal Pro Trp Lys Gly 115 120 125 Thr His Ser Leu Thr Lys Phe Ser Cys LysAsn Ala 130 135 140 6 146 PRT Homo sapiens 6 Met Ala Gly Pro Leu Arg AlaPro Leu Leu Leu Leu Ala Ile Leu Ala 1 5 10 15 Val Ala Leu Ala Val SerPro Ala Ala Gly Ser Ser Pro Gly Lys Pro 20 25 30 Pro Arg Leu Val Gly GlyPro Met Asp Ala Ser Val Glu Glu Glu Gly 35 40 45 Val Arg Arg Ala Leu AspPhe Ala Val Gly Glu Tyr Asn Lys Ala Ser 50 55 60 Asn Asp Met Tyr His SerArg Ala Leu Gln Val Val Arg Ala Arg Lys 65 70 75 80 Gln Ile Val Ala GlyVal Asn Tyr Phe Leu Asp Val Glu Leu Gly Arg 85 90 95 Thr Thr Cys Thr LysThr Gln Pro Asn Leu Asp Asn Cys Pro Phe His 100 105 110 Asp Gln Pro HisLeu Lys Arg Lys Ala Phe Cys Ser Phe Gln Ile Tyr 115 120 125 Ala Val ProTrp Gln Gly Thr Met Thr Leu Ser Lys Ser Thr Cys Gln 130 135 140 Asp Ala145 7 142 PRT Homo sapiens 7 Met Met Trp Pro Met His Thr Pro Leu Leu LeuLeu Thr Ala Leu Met 1 5 10 15 Val Ala Val Ala Gly Ser Ala Ser Ala GlnSer Arg Thr Leu Ala Gly 20 25 30 Gly Ile His Ala Thr Asp Leu Asn Asp LysSer Val Gln Arg Ala Leu 35 40 45 Asp Phe Ala Ile Ser Glu Tyr Asn Lys ValIle Asn Lys Asp Glu Tyr 50 55 60 Tyr Ser Arg Pro Leu Gln Val Met Ala AlaTyr Gln Gln Ile Val Gly 65 70 75 80 Gly Val Asn Tyr Tyr Phe Asn Val LysPhe Gly Arg Thr Thr Cys Thr 85 90 95 Lys Ser Gln Pro Asn Leu Asp Asn CysPro Phe Asn Asp Gln Pro Lys 100 105 110 Leu Lys Glu Glu Glu Phe Cys SerPhe Gln Ile Asn Glu Val Pro Trp 115 120 125 Glu Asp Lys Ile Ser Ile LeuAsn Tyr Lys Cys Arg Lys Val 130 135 140 8 149 PRT Homo sapiens 8 Met AlaArg Ser Asn Leu Pro Leu Ala Leu Gly Leu Ala Leu Val Ala 1 5 10 15 PheCys Leu Leu Ala Leu Pro Arg Asp Ala Arg Ala Arg Pro Gln Glu 20 25 30 ArgMet Val Gly Glu Leu Arg Asp Leu Ser Pro Asp Asp Pro Gln Val 35 40 45 GlnLys Ala Ala Gln Ala Ala Val Ala Ser Tyr Asn Met Gly Ser Asn 50 55 60 SerIle Tyr Tyr Phe Arg Asp Thr His Ile Ile Lys Ala Gln Ser Gln 65 70 75 80Leu Val Ala Gly Ile Lys Tyr Phe Leu Thr Met Glu Met Gly Ser Thr 85 90 95Asp Cys Arg Lys Thr Arg Val Thr Gly Asp His Val Asp Leu Thr Thr 100 105110 Cys Pro Leu Ala Ala Gly Ala Gln Gln Glu Lys Leu Arg Cys Asp Phe 115120 125 Glu Val Leu Val Val Pro Trp Gln Asn Ser Ser Gln Leu Leu Lys His130 135 140 Asn Cys Val Gln Met 145 9 145 PRT Homo sapiens 9 Met Arg AlaAla Gly Thr Leu Leu Ala Phe Cys Cys Leu Val Leu Ser 1 5 10 15 Thr ThrGly Gly Pro Ser Pro Asp Thr Cys Ser Gln Asp Leu Asn Ser 20 25 30 Arg ValLys Pro Gly Phe Pro Lys Thr Ile Lys Thr Asn Asp Pro Gly 35 40 45 Val LeuGln Ala Ala Arg Tyr Ser Val Glu Lys Phe Asn Asn Cys Thr 50 55 60 Asn AspMet Phe Leu Phe Lys Glu Ser Arg Ile Thr Arg Ala Leu Val 65 70 75 80 GlnIle Val Lys Gly Leu Lys Tyr Met Leu Glu Val Glu Ile Gly Arg 85 90 95 ThrThr Cys Lys Lys Asn Gln His Leu Arg Leu Asp Asp Cys Asp Phe 100 105 110Gln Thr Asn His Thr Leu Lys Gln Thr Leu Ser Cys Tyr Ser Glu Val 115 120125 Trp Val Val Pro Trp Leu Gln His Phe Glu Val Pro Val Leu Arg Cys 130135 140 His 145 10 149 PRT Homo sapiens 10 Met Ala Arg Ser Asn Leu ProLeu Ala Leu Gly Leu Ala Leu Val Ala 1 5 10 15 Phe Cys Leu Leu Ala LeuPro Arg Asp Ala Arg Ala Arg Pro Gln Glu 20 25 30 Arg Met Val Gly Glu LeuArg Asp Leu Ser Pro Asp Asp Pro Gln Val 35 40 45 Gln Lys Ala Ala Gln AlaAla Val Ala Ser Tyr Asn Met Gly Ser Asn 50 55 60 Ser Ile Tyr Tyr Phe ArgAsp Thr His Ile Ile Lys Ala Gln Ser Gln 65 70 75 80 Leu Val Ala Gly IleLys Tyr Phe Leu Thr Met Glu Met Gly Ser Thr 85 90 95 Asp Cys Arg Lys ThrArg Val Thr Gly Asp His Val Asp Leu Thr Thr 100 105 110 Cys Pro Leu AlaAla Gly Ala Gln Gln Glu Lys Leu Arg Cys Asp Phe 115 120 125 Glu Val LeuVal Val Pro Trp Gln Asn Ser Ser Gln Leu Leu Lys His 130 135 140 Asn CysVal Gln Met 145 11 141 PRT Homo sapiens 11 Met Ala Arg Pro Leu Cys ThrLeu Leu Leu Leu Met Ala Thr Leu Ala 1 5 10 15 Gly Ala Leu Ala Ser SerSer Lys Glu Glu Asn Arg Ile Ile Pro Gly 20 25 30 Gly Ile Tyr Asp Ala AspLeu Asn Asp Glu Trp Val Gln Arg Ala Leu 35 40 45 His Phe Ala Ile Ser GluTyr Asn Lys Ala Thr Glu Asp Glu Tyr Tyr 50 55 60 Arg Arg Pro Leu Gln ValLeu Arg Ala Arg Glu Gln Thr Phe Gly Gly 65 70 75 80 Val Asn Tyr Phe PheAsp Val Glu Val Gly Arg Thr Ile Cys Thr Lys 85 90 95 Ser Gln Pro Asn LeuAsp Thr Cys Ala Phe His Glu Gln Pro Glu Leu 100 105 110 Gln Lys Lys GlnLeu Cys Ser Phe Glu Ile Tyr Glu Val Pro Trp Glu 115 120 125 Asp Arg MetSer Leu Val Asn Ser Arg Cys Gln Glu Ala 130 135 140 12 141 PRT Homosapiens 12 Met Ala Gln His Leu Ser Thr Leu Leu Leu Leu Leu Ala Thr LeuAla 1 5 10 15 Val Ala Leu Ala Trp Ser Pro Lys Glu Glu Asp Arg Ile IlePro Gly 20 25 30 Gly Ile Tyr Asn Ala Asp Leu Asn Asp Glu Trp Val Gln ArgAla Leu 35 40 45 His Phe Ala Ile Ser Glu Tyr Asn Lys Ala Thr Lys Asp AspTyr Tyr 50 55 60 Arg Arg Pro Leu Arg Val Leu Arg Ala Arg Gln Gln Thr ValGly Gly 65 70 75 80 Val Asn Tyr Phe Phe Asp Val Glu Val Gly Arg Thr IleCys Thr Lys 85 90 95 Ser Gln Pro Asn Leu Asp Thr Cys Ala Phe His Glu GlnPro Glu Leu 100 105 110 Gln Lys Lys Gln Leu Cys Ser Phe Glu Ile Tyr GluVal Pro Trp Glu 115 120 125 Asn Arg Arg Ser Leu Val Lys Ser Arg Cys GlnGlu Ser 130 135 140 13 68 PRT Artificial Sequence ZCYS3 motif 13 Gln XaaXaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 XaaXaa Cys Xaa Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 XaaCys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 45 XaaXaa Xaa Xaa Xaa Xaa Xaa Pro Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 XaaXaa Xaa Cys 65 14 46 PRT Artificial Sequence Cysteine motif 14 Cys XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa 1 5 10 15 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 45 15 423 DNAArtificial Sequence Degenerate nucleotide sequence encoding the zcys3polypeptide of SEQ ID NO2 15 atggcnmgnt tyytncarac nytnytntty ytngtnathacngtngartt ygtnwsnmgn 60 mgngtngarg cntggggnws nccncarath gtnmgnccnttygargayat hccnaarwsn 120 taygtntayg tncarcaygc nytntggtay gcnatgaargartayaayaa rgcnwsnaay 180 gayytntaya ayttymgngt ngtngayath ytnaarwsncargarcarat hacngaywsn 240 ytngartayt ayytngargt naayathgcn mgnacnatgtgyaaraarat hgcnggngay 300 aaygaraayt gyytnttyca rcargayccn aaratgaaraaratggtntt ytgyathtty 360 athgtnwsnw snaarccntg gaarttygar ytnaaratgytnaaraarca rtgyaargay 420 ath 423 16 20 DNA Artificial SequenceOligonucleotide ZC18696 16 ctacattgac accagctctg 20 17 20 DNA ArtificialSequence Oligonucleotide ZC18369 17 aaggtgtccc gaatgctgat 20 18 137 PRTMus musculus 18 Met Ser Cys Pro Leu Arg Lys Lys Ala Leu Pro Leu Thr MetLeu Leu 1 5 10 15 Leu Leu Leu Ser Phe His Val Leu Ile Thr Pro Val SerLys Ala Asn 20 25 30 Lys Glu Thr Asn Arg Ser Val His Phe Ile Pro Thr ValGlu Phe Ala 35 40 45 Val Asn Thr Phe Asn Gln Glu Ser Gln Asp Glu Tyr AlaTyr Arg Met 50 55 60 Glu His Ile Met Ser Ser Trp Arg Glu Lys Val Asn PhePro Thr Val 65 70 75 80 Tyr Ser Met Arg Leu Gln Leu Arg Arg Thr Ile CysLys Lys Phe Glu 85 90 95 Glu Ser Leu Asp Ile Cys Pro Phe Gln Glu Ser HisGly Leu Asn Asn 100 105 110 Thr Phe Thr Cys Leu Phe Thr Val Gly Thr TyrPro Trp Ile Thr Lys 115 120 125 Phe Lys Leu Phe Arg Ser Val Cys Ser 130135 19 18 DNA Artificial Sequence Oligonucleotide ZC20814 19 cacggtggagtttgtatc 18 20 18 DNA Artificial Sequence Oligonucleotide ZC20815 20gctgcacata gacatagg 18 21 23 DNA Artificial Sequence OligonucleotideZC17516 21 aagtaagagt ggcaaggtgt ccc 23 22 22 DNA Artificial SequenceOligonucleotide ZC17517 22 gcccgaacaa tgtgcaagaa ga 22

What is claimed is:
 1. An isolated polynucleotide encoding a polypeptidecomprising 10 or more contiguous amino acid residues of SEQ ID NO: 2,wherein said polypeptide comprises SEQ ID NO: 14 and inhibits cysteineproteinase.
 2. An isolated polynucleotide according to claim 1, whereinsaid polypeptide comprises SEQ ID NO:13.
 3. An isolated polynucleotideaccording to claim 1, wherein said polypeptide comprises amino acidresidues 76-138 of SEQ ID NO:2.
 4. An isolated polynucleotide thatencodes a polypeptide which inhibits cysteine proteinases and hybridizesunder stringent conditions to SEQ ID NO: 1, wherein said stringenthybridization conditions comprise hybridizing in 6×SSC at about 65° C.and washing in 0.1×SSC at about 65° C.
 5. An isolated polynucleotidemolecule consisting of nucleotides 271-459 of SEQ ID NO:1.
 6. Anisolated polynucleotide that encodes a polypeptide which inhibitscysteine proteinases, wherein said polynucleotide comprises thenucleotide sequence of nucleotides 46 to 468 of SEQ ID NO:1.
 7. Anisolated polynucleotide according to claim 1, further comprising a DNAsegment encoding an affinity tag or binding domain.
 8. A polynucleotideencoding a fusion protein comprising a secretory signal sequence havingthe amino acid sequence of amino acid residues 1-20 of SEQ ID NO:2,wherein said secretory signal sequence is fused to the N-terminus of apolypeptide.
 9. A polynucleotide molecule encoding a fusion proteinconsisting of a first portion and a second portion joined by a peptidebond, said first portion comprising a polypeptide having 10 or morecontiguous amino acid residues of SEQ ID NO: 2, wherein said polypeptidecomprises SEQ ID NO: 14 and inhibits cysteine proteinases; and saidsecond portion comprising another polypeptide.
 10. An expression vectorcomprising the following operably linked elements: a transcriptionpromoter; a DNA segment encoding a polypeptide comprising 10 or morecontiguous amino acid residues of SEQ ID NO: 2, wherein said polypeptidecomprises SEQ ID NO: 14 and inhibits cysteine proteinases; and atranscription terminator.
 11. An expression vector according to claim 10further comprising a DNA segment encoding a secretory signal sequencefused to the N-terminus of said polypeptide encoded by said DNA segment.12. An expression vector according to claim 10, wherein said DNA segmentencodes a polypeptide covalently linked amino terminally or carboxyterminally to an affinity tag.
 13. A cultured cell into which has beenintroduced an expression vector according the claim 10, wherein saidcultured cell expresses said polypeptide encoded by said DNA segment.14. A method of producing a polypeptide comprising: culturing a cellinto which has been introduced an expression vector according to claim10; whereby said cell expresses said polypeptide encoded by said DNAsegment; and recovering said expressed polypeptide.